U.S. patent application number 12/402352 was filed with the patent office on 2009-11-19 for clostridium botulinum c3 exotransferase compositions and methods for treating tumour spreading.
This patent application is currently assigned to BIOAXONE THERAPEUTIQUE INC.. Invention is credited to Dana Lasko, Lisa McKerracher.
Application Number | 20090285833 12/402352 |
Document ID | / |
Family ID | 34396295 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090285833 |
Kind Code |
A1 |
McKerracher; Lisa ; et
al. |
November 19, 2009 |
CLOSTRIDIUM BOTULINUM C3 EXOTRANSFERASE COMPOSITIONS AND METHODS
FOR TREATING TUMOUR SPREADING
Abstract
Pharmaceutical compositions, each consisting of a cell-permeable
fusion protein conjugate of a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, are provided. The compositions are
useful to prevent or inhibit uncontrolled proliferation, spreading,
and migration of a metastatic neoplastic cell of a cancer in a
mammal. The compositions can each effect or arrest combination of
two or more of tumor cell proliferation, migration, angiogenesis,
and metalloproteinase secretion.
Inventors: |
McKerracher; Lisa; (Ile des
Soeurs, CA) ; Lasko; Dana; (Montreal, CA) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
BIOAXONE THERAPEUTIQUE INC.
Saint Laurent
CA
|
Family ID: |
34396295 |
Appl. No.: |
12/402352 |
Filed: |
March 11, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10573658 |
Nov 7, 2006 |
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PCT/CA04/01763 |
Sep 29, 2004 |
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12402352 |
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60506162 |
Sep 29, 2003 |
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Current U.S.
Class: |
424/167.1 |
Current CPC
Class: |
A61K 9/0024 20130101;
A61P 35/04 20180101; C07K 2319/00 20130101; C12N 9/1077 20130101;
A61K 47/64 20170801; A61K 47/6415 20170801; C07K 14/43563 20130101;
A61K 9/19 20130101; A61K 47/6425 20170801; Y02A 50/469 20180101;
A61K 38/45 20130101; A61K 47/6829 20170801; Y02A 50/30 20180101;
A61P 35/00 20180101; A61K 38/164 20130101 |
Class at
Publication: |
424/167.1 |
International
Class: |
A61K 39/40 20060101
A61K039/40 |
Claims
1. A method of prevention or inhibition of uncontrolled
proliferation and spreading or migration of a metastatic neoplastic
cell of a cancer in a mammal, comprising administration to the
mammal of a therapeutically effective amount of a pharmaceutical
composition comprising a cell-permeable fusion protein conjugate
comprising a polypeptidic cell-membrane transport moiety and a
Clostridium botulinum C3 exotransferase unit, or a functional
analog thereof.
2. A method of prevention or inhibition of uncontrolled
proliferation and spreading or migration, within a resection margin
of a host tissue proximal to the site of excision of a tumor of a
cancer in a mammal, of a metastatic neoplastic cell residing in the
resection margin, comprising administration of a therapeutically
effective amount of a pharmaceutical composition comprising a
cell-permeable fusion protein conjugate comprising a polypeptidic
cell-membrane transport moiety and a Clostridium botulinum C3
exotransferase unit, or a functional analog thereof, said
administration being directly on to the surface of the resection
margin or below the surface of the resection margin or into the
tissue proximal to the resection margin which remains in the
mammal, said administration in a time interval prior to or
subsequent to or prior to and subsequent to excision or removal of
the tumor.
3. A method of prevention of growth of a tumor from a malignant
cell in a host tissue in a mammal comprising administration to the
mammal of a therapeutically effect amount of a pharmaceutical
composition comprising a cell-permeable fusion protein conjugate
comprising a polypeptidic cell-membrane transport moiety and a
Clostridium botulinum C3 exotransferase unit, or a functional
analog thereof, wherein the fusion protein simultaneously prevents
or inhibits at least two of malignant cell migration, malignant
cell proliferation, angiogenesis or tubular structure formation or
capillary network growth proximal to the malignant cell, and
secretion of an active metalloproteinase from the malignant
cell.
4. A method of prevention of growth within a resection margin of a
host tissue proximal to a site of excision or removal of a first
tumor of a cancer in a mammal, of a second tumor comprising a
residual tumor cell of the cancer, the method comprising
administration of a therapeutically effective amount of a
pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, said administration being directly on to
the surface of the resection margin or below the surface of the
resection margin or into the tissue proximal to the resection
margin which remains in the mammal, and said administration being
in a time interval prior to, or subsequent to, or both prior to and
subsequent to excision or removal of the first tumor, wherein the
fusion protein simultaneously prevents or inhibits at least two of
residual tumor cell migration, residual tumor cell proliferation,
angiogenesis or tubular structure formation or capillary network
growth proximal to the residual tumor cell, and secretion of an
active metalloproteinase from the residual tumor cell.
5. The method of claim 1, wherein the fusion protein conjugate has
SEQ ID NO:4.
6. The method of claim 1, wherein the cancer is selected from the
group consisting of breast, brain, colon, skin, kidney, and hepatic
cancer.
7. The method of claim 1, wherein the cancer is a brain tumor
selected from the group consisting of glial tumors, neuron tumors,
pineal gland tumors, menigeal tumors, tumors of nerve sheath,
lymphomas, malformative tumors, and metastatic tumors located in
the brain derived from tumors of the lung, breast, melanoma,
kidney, and gastrointestinal tract.
8. The method of claim 1, wherein the cancer is a brain tumor
selected from the group consisting of anaplastic astrocytoma,
glioblastoma multiform, pilocytic astrocytoma, oligodendroglioma,
ependymoma, myxopapillary ependymoma, subependymoma, choroid plexus
papilloma, neuroblastoma, ganglioneuroblastoma, ganglioneuroma, and
medulloblastoma, pineoblastoma and pineocytoma, meningioma,
meningeal hemangiopericytoma, meningeal sarcoma, Schwannoma
(neurolemmoma) and neurofibroma, Hodgkin's lymphoma, non-Hodgkin's
lymphoma, primary and secondary subtypes of Hodgkin's lymphoma,
primary and secondary subtypes of non-Hodgkin's lymphoma,
craniopharyngioma, epidermoid cysts, dermoid cysts and colloid
cysts.
9. The method of claim 1, wherein the therapeutically effective
amount is about 0.001 micrograms per cc to about 50 micrograms per
cc of tissue.
10. The method of claim 1, wherein the therapeutically effective
amount is about 0.0001 micrograms of fusion protein per cubic
centimeter (cc) of tissue to about 100 micrograms per cubic
centimeter of tissue.
11. The method of claim 1, wherein the therapeutically effective
amount is about 1 microgram per milliliter to about 10 micrograms
per milliliter to about 50 micrograms per milliliter.
12. The method of claim 1, wherein the administration is by
injection, by topical application, or by implantation.
13. The method of claim 1, wherein the administration is selected
from the group consisting of intrarticular, intraocular,
intranasal, intraneural, intradermal, intraosteal, sublingual,
oral, topical, intravesical, intrathecal, intravenous,
intraperitoneal, intracranial, intramuscular, subcutaneous,
inhalation, atomization and inhalation, application directly into a
tumor, application directly into a disease site, application
directly on or into the margins remaining after resection of a
tumor, enteral, enteral together with a gastroscopic procedure, and
ECRP.
14. The method of claim 1, wherein the polypeptidic cell-membrane
transport moiety comprises a peptide containing from about 5 to
about 50 amino acids.
15. The method of claim 1, wherein the Clostridium botulinum C3
exotransferase unit comprises the amino acid sequence designated by
the sequence of fusion protein BA-05.
16. The method of claim 1, wherein the functional analog comprises
a protein exhibiting activity in the range of 50% to 500% of that
of wild type Clostridium botulinum C3 exotransferase.
17. The method of claim 1, wherein the pharmaceutical composition
comprises a pharmaceutically acceptable carrier.
18. The method of claim 1, wherein the pharmaceutical composition
comprises a pharmaceutically acceptable carrier selected from the
group consisting of poly(ethylene-co-vinyl acetate), PVA, partially
hydrolyzed poly(ethylene-co-vinyl acetate), poly(ethylene-co-vinyl
acetate-co-vinyl alcohol), a cross-linked poly(ethylene-co-vinyl
acetate), a cross-linked partially hydrolyzed
poly(ethylene-co-vinyl acetate), a cross-linked
poly(ethylene-co-vinyl acetate-co-vinyl alcohol), poly-D,L-lactic
acid, poly-L-lactic acid, polyglycolic acid, PGA, copolymers of
lactic acid and glycolic acid, polycaprolactone, polyvalerolactone,
poly (anhydrides), copolymers of polycaprolactone with polyethylene
glycol, copolymers of polylactic acid with polyethylene glycol,
polyethylene glycol; and combinations and blends thereof.
19. The method of claim 1, wherein the pharmaceutical composition
comprises a pharmaceutically acceptable carrier comprising an
aqueous gelatin, an aqueous protein, a polymeric carrier, a
cross-linking agent, and a combination thereof.
20. The method of claim 1, wherein the pharmaceutical composition
comprises a pharmaceutically acceptable carrier comprising a
matrix.
21. The method of claim 1, wherein the pharmaceutical composition
comprises a pharmaceutically acceptable carrier comprising water, a
pharmaceutically acceptable buffer salt, a pharmaceutically
acceptable buffer solution, a pharmaceutically acceptable
antioxidant, ascorbic acid, one or more low molecular weight
pharmaceutically acceptable polypeptide, a peptide comprising about
2 to about 10 amino acid residues, one or more pharmaceutically
acceptable protein, one or more pharmaceutically acceptable amino
acid, an essential-to-human amino acid, one or more
pharmaceutically acceptable carbohydrate, one or more
pharmaceutically acceptable carbohydrate-derived material, a
non-reducing sugar, glucose, sucrose, sorbitol, trehalose,
mannitol, maltodextrin, dextrins, cyclodextrin, a pharmaceutically
acceptable chelating agent, EDTA, DTPA, a chelating agent for a
divalent metal ion, a chelating agent for a trivalent metal ion,
glutathione, pharmaceutically acceptable nonspecific serum albumin,
and combinations thereof.
22. The method of claim 1, wherein the pharmaceutical composition
is sterile.
23. The method of claim 1, wherein the pharmaceutical composition
is sterilizable.
24. The method of claim 1, wherein the pharmaceutical composition
is sterilized.
25. The method of claim 1, wherein the pharmaceutical composition
is in a vial in a unit dosage amount or in an integral multiple of
a unit dosage amount.
26. The method of claim 1, wherein the pharmaceutical composition
is dried.
27. The method of claim 1, wherein the pharmaceutical composition
comprises a dehydrated matrix.
28. The method of claim 1, wherein the pharmaceutical composition
comprises a pharmaceutically acceptable carrier.
29. The method of claim 1, wherein the pharmaceutical composition
comprises a fusion protein in a lyophilized matrix.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/573,658 filed Mar. 28, 2006 which is a continuation of U.S.
application Ser. No. 10/902,878, both of which claim priority under
35 U.S.C. .sctn. 119 to PCT/CA2004/001763, filed Sep. 29, 2004 and
also claim priority under 35 U.S.C. .sctn. 120 to U.S. Provisional
application 60/506,162, filed Sep. 29, 2003, all of which are
herein incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods
useful for the treatment of cancer and the prevention of tumor
growth related to metastatic cancer. In particular, the present
invention relates to compositions comprising a cell-permeable
fusion protein conjugate comprising a polypeptidic cell-membrane
transport moiety and a Clostridium botulinum C3 exotransferase
unit, or a functional analog thereof useful for prevention or
inhibition of uncontrolled proliferation and spreading or migration
of a metastatic neoplastic cell of a cancer in a mammal.
BACKGROUND
[0003] Cancer in a mammal can be characterized by the uncontrolled
division of a population of malignant cells within a tissue in the
mammal. If the cell population is localized in a tissue, such
uncontrolled division of malignant or cancer cells can lead to the
formation of a malignant first tumor in the tissue. If one or more
malignant cell or cluster of cells migrates from the site of the
localized population to lodge or take root and grow uncontrolled in
a second site or in additional tissue sites, which site or sites
may be proximal to the first tumor site or which may be remote from
the first tumor site, for example in another organ or tissue
anatomically distant or distinct from the first tissue, then a
second tumor or additional tumors can emerge at the second site or
additional sites, respectively, as a result of uncontrolled
division of the migrated malignant cell or cells. Migration of one
or more malignant cells from the locus of growing cells at the
second site to other sites can also occur, and so forth to produce
malignant tumors at one or more tissue sites in the mammal.
Associated with the growth of such malignant tumors is an often
characteristic angiogenesis or process of vascularisation of a
tissue proximal to the evolving tumor comprising the development of
new capillary blood vessels or in-growth of vasculature and tubular
network formation, which new vasculature provides various factors
such as nutrients and growth factors that are necessary and permit
continued tumor growth.
[0004] A tumor is an abnormal mass of tissue that results from
excessive cell division that is uncontrolled and progressive, also
called a neoplasm. Tumors may be either benign (not cancerous) or
malignant.
[0005] A variety of methods are presently utilized to treat cancer
in a mammal such as man, including for example, surgical procedures
in which, for example a tumor and usually some contiguous or
proximal non-tumorous tissue is excised from the site of the tumor
in a tissue. After removal of the tumor, residual or marginal
tissue remains proximal to the site of excision of the tumor in the
mammal. If treated with surgery alone however, many patients,
particularly those with certain types of cancer, such as cancer
selected from the group consisting of breast, brain, colon, skin
(melanoma), kidney (renal) and hepatic (liver) cancer will
experience recurrence of the cancer in the form of the formation
and growth of at least one additional or second tumor, often in the
residual margins remaining after excision of the first tumor and
sometimes in other tissue or organs and in locations remote or
distant from the site of the first tumor. Therefore, in addition to
surgery, many cancers are also treated with a combination of
therapies, such as those involving administration of cytotoxic
chemotherapeutic drugs (e.g., vincristine, vinblastine, cisplatin,
methotrexate, 5-FU, etc.) and/or radiation therapy. One difficulty
with this approach, however, is that radiotherapeutic and
chemotherapeutic agents can be toxic to normal tissues at the dose
levels administered, and often create life-threatening side effects
in the patient. These cancer therapies can often have high
failure/remission rates which can result in death of the patient.
Some more recent therapeutic treatments take advantage of
dysregulation of cellular signaling by altered or upregulated gene
products in cancer cells, such a the use of tamoxifen for breast
cancer and Gleevec.RTM. (imatinib mesylate from Novartis) for
chronic myeloid leukemia (also referred to as CML).
[0006] An additional difficulty of present methods is that local
recurrence and local disease control remains a major challenge in
the treatment of malignancy. Over 600,000 patients annually (in the
U.S.) have localized malignant disease (with no evidence of distant
metastatic spread) at the time of presentation, representing about
64% of all patients diagnosed with malignancy but not including
nonmelanoma skin cancer or carcinoma in situ. For a majority of
these patients, surgical resection of the disease represents the
greatest chance for a cure, and over 400,000 patients will be cured
after the initial treatment. Unfortunately, about 200,000 (or about
one third of all patients with localized disease) will relapse
after the initial treatment. Of those who relapse, the number who
will relapse due to local recurrence of the disease can amount to
about 133,000 patients annually (or about 21% of all those with
localized disease). The number who will relapse due to distant
metastases of the disease is about 68,000 patients annually (or
about 11% of all those with localized disease). About another
100,000 patients annually will die as a direct result of an
inability to control the local growth of the disease.
[0007] Brain tumors are an especially deadly form of cancer. About
one third of all primary gliomas (gliomas represent about 1/3 of
all brain tumors) are fatal, and the mean survival for glioma
patients is about 10 to about 12 months. The five year survival
rate is about 9%. Gliomas are neuroectodermal tumors of neuroglial
origin, and include astrocytoma derived from astrocytes,
oligodendroglioma derived from oligodendrocytes, and ependymoma
derived from ependymal cells. A number of studies suggest that
combination therapies will needed to treat these aggressive tumors.
The most common type of brain tumor arises by metastasis, and there
are about 100,000 to about 170,000 brain tumors diagnosed per year
in the USA. The mean survival time ranges from about 2.9 months to
about 3.4 months. Metastatic brain tumors are mainly treated with
radiosurgery or tumor resection. Better outcomes have been reported
when surgery is combined with radiation than with radiation alone.
The most common origins for metastatic tumors to the brain comprise
mammary cancers, bronchial cancers, gastrointestinal carcinoma,
renal carcinoma, and malignant melanoma. Metastatic brain tumors
may be may be clinically explosive, especially after removal of a
primary tumor. Individuals suspected of having CNS cancer (which
includes brain tumors and brain cancer as used herein) may be
identified by detecting clinical symptoms such as headache, nausea
or vomiting, seizures, altered mental status, altered speech,
visual abnormalities, and/or paralysis. A method of inhibiting
metastases of a primary CNS cancer in a mammal is also within the
scope of the present invention.
Angiogensis
[0008] Many of the mechanisms which control angiogenesis in normal
tissues are altered in the presence of a malignant tumors during
tumor growth. The formation and metastasis of a tumor involved
pathological angiogenesis. Like healthy tissues, a tumor requires
connection to blood vessels in order to receive nutrients and
oxygen and to eliminate cellular wastes. Thus, pathological
angiogenesis is critical to the growth and expansion of tumors.
Tumors in which angiogenesis is important include solid malignant
tumors as well as benign tumors, for example such as acoustic
neuroma, neurofibroma, trachoma and pyogenic granulomas. In
metastasis, pathological angiogenesis is important in at least two
aspects. The formation of blood vessels in tumors allows tumor
cells to enter the blood stream and to circulate throughout the
body. Angiogenesis supports the formation and growth of new tumors
seeded by tumor cells that have left the primary site or first
tumor as used herein.
[0009] Angiogenesis is the complex process of blood vessel
formation. The process involves both biochemical and cellular
events, including (1) activation of endothelial cells (ECs) by an
angiogenic stimulus; (2) degradation of the extracellular matrix,
invasion of the activated ECs into the surrounding tissues, and
migration toward the source of the angiogenic stimulus; and (3)
proliferation and differentiation of ECs to form new blood vessels
(Folkman et al., 1991, J. Biol. Chem. 267:10931-10934).
[0010] The control of angiogenesis is a highly regulated process
involving angiogenic stimulators and inhibitors. In healthy humans
and animals, angiogenesis occurs under specific, restricted
situations. For example, angiogenesis is normally observed in fetal
and embryonal development, development and growth of normal tissues
and organs, wound healing, and the formation of the corpus luteum,
endometrium and placenta.
[0011] Another embodiment of the present invention comprises the
inhibition of angiogenesis by a cell-permeable fusion protein
conjugate comprising a polypeptidic cell-membrane transport moiety
and a Clostridium botulinum C3 exotransferase unit, or a functional
analog thereof, for example a fusion protein such as BA-05.
[0012] Another embodiment of the present invention comprises the
inhibition of angiogenesis by an effective amount of a
pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, for example a fusion protein such as
BA-05.
Rho Signaling and Cancer
[0013] Rho (also known as Ras homology) family proteins have been
investigated in relation to cancer. Ras (and RhoB as a secondary
target) are targets for metastasis by molecules that inhibit
posttranslational modification. However, these therapeutics
investigations focus on Ras and are limited to RhoB among Rho
family members, whereas the current invention has the potential to
affect signaling of RhoA, RhoB, and RhoC. RhoA, RhoB and Rho C are
Rho family members specifically inhibited by the fusion protein
BA-05. In some studies, C3 exoenzyme has been used as a molecular
probe for Rho involvement and significant changes have been found
in parameters of in vitro models considered important in cancer
such as cell transformation. In such studies C3 was applied by
methods ranging from prolonged incubation in the tissue culture
medium to heterologous gene expression. It is an advantage of the
current invention that compositions and methods of the current
invention such as BA-05 and administration of BA-05 offer a
significant advantage compared to C3 because of the ability of
compositions of the current invention to penetrate inside tumor
cells to inactivate rapidly Rho at lower doses. In another
advantage, the current invention provides compositions comprising a
fusion protein of this invention such as BA-07, which fusion
protein has the ability to penetrate both tumour cells and
endothelial cells that in the absence of the fusion protein can
form new blood vessels that supply tumor growth.
[0014] Mutations in Rho family regulatory proteins have been found
in clinical samples of malignancies. Examples include the DLC1 gene
in hepatocellular carcinoma; p-190-A, in a genomic region that is
altered in gliomas and astrocytomas; GRAF, which has loss of
function mutations in leukemia; and LARG, found in some a gene
fusions found in acute myeloid leukemia. Genetically engineered
point mutations activate RhoA and induce cellular transformation in
vitro.
Rho Family Gene Expression in Human Malignancy
[0015] The small GTPase Rho is a cellular target of BA-05, and is
up-regulated in certain cancers, such as malignant melanoma and
breast cancer.
[0016] In contrast to the small GTPase Ras, Rho GTPases have not
been identified as oncogenes by traditional approaches, although
evidence has accumulated for dysregulation of Rho gene expression
in cancer. For instance, increased levels of RhoA mRNA have been
observed in testicular germ cell tumor, and increased RhoC mRNA in
inflammatory breast cancer and pancreatic adenocarcinoma.
[0017] The Cancer Genome Anatomy Project (CGAP) correlates gene
expression with site of malignancy. Data is available on
transcription levels in libraries made from malignant and normal
cells (NCBI, 2002). Transcription levels are measured using "tags",
i.e., 10 base oligonucleotides that uniquely define a gene.
Available data on RhoA, RhoB and RhoC I shows upregulation of RhoA
and to a lesser extent in these measurements, RhoC in malignancies
of the brain and in the breast. Rho A sequence tags are found more
often in libraries made from malignancies of the cerebellum and
breast than from the corresponding normal tissue. Expression levels
were elevated in glioblastoma but not in astrocytoma. The result
for astrocytoma corresponds with reduction in RhoA protein levels
in astrocytic tumor samples. Rho C mRNA is overexpressed in breast
malignancies and to a slight extent in some brain malignancies, and
may be downregulated in colon adenocarcinoma.
[0018] However, relative levels of Rho cDNA in such libraries may
not directly relate to Rho action in the cell, which undergoes
complex regulation involving numerous other gene products.
Rho Proteins in Tumors and Tumor Cell Lines
[0019] Rho protein expression has been investigated at several
tumor sites in humans. Increased protein levels are found in colon,
breast and lung tumors. RhoA and RhoB levels have been found in 5
.mu.m sections from head and neck squamous cell carcinomas using
polyclonal antibodies directed against these proteins, followed by
visualization using a VectaStain kit (Vector Labs) and image
analysis. Nearby "normeoplastic" areas were used as controls.
Although Rho A protein levels increased with tumor progression,
RhoB levels decreased in invasive tumors compared to carcinomas in
situ and well-differentiated tumors. Activation states were not
studied.
[0020] Overexpression of RhoA and RhoB may occur in breast and lung
adenocarcinomas compared to normal tissue, whereas expression of
Rho proteins is decreased in astrocytic tumors and inversely
related to grade II to IV malignancy.
Rho and Metastasis
[0021] Rho is involved in regulation of cell migration and
motility. MM1 rat hepatoma cells transfected with Rho A mutant
constructs (Val.sup.14 or Val.sup.14Ile.sup.41) result in
constitutively activated Rho. In an in vitro invasion assay, the
percent of seeded cells capable of infiltration into a mesothelial
cell layer was correlated with the level of expression of
transfected RhoA Val.sup.14. When these activated RhoA-transfected
cells were used in an in vivo assay in the peritoneal cavity, 6 of
10 implants resulted in tumor nodules compared with 2 of 8 for mock
transfectants. These results indicate that active Rho is correlated
with tumorigenicity.
[0022] A comprehensive study of gene expression compared two
metastatic melanoma model systems, one human and one mouse, and
looked at the shared similarities in gene expression by microarray
concluded that RhoC expression was altered in increasing levels of
metastasis (Clark et al., 2000). Furthermore, when gene expression
was manipulated experimentally, RhoC overexpression induced a human
melanoma cell line to switch from low metastatic potential to high
metastatic potential. Although RhoA was not observed to be
overexpressed, a dominant negative mutation (N19RhoA) diminished
metastatic potential.
[0023] A set of 70 genes whose expression correlated with
propensity for metastasis in human breast cancer was identified
(van't Veer et al., 2002). Although Rho genes were not found, the
value of a disease marker as a prognostic indicator is not
necessarily related to its value as a target for therapy. In the
case of Rho family signaling, there is complex regulation of
enzymatic activity and protein-protein interactions which is not
apparent from measurements of transcription levels alone.
SUMMARY OF THE INVENTION
[0024] Individual fusion proteins of this invention are sometimes
referred to by designations such as BA-05, BA-07, and the like.
[0025] This invention discloses a method of prevention or
inhibition of uncontrolled proliferation and spreading or migration
of a metastatic neoplastic cell of a cancer in a mammal, comprising
administration to the mammal of a therapeutically effective amount
of a pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof.
[0026] This invention discloses a method of prevention or
inhibition of uncontrolled proliferation and spreading or
migration, within a resection margin of a host tissue proximal to
the site of excision of a tumor of a cancer in a mammal, of a
metastatic neoplastic cell residing in the resection margin,
comprising administration of a therapeutically effective amount of
a pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, said administration being directly on to
the surface of the resection margin or below the surface of the
resection margin or into the tissue proximal to the resection
margin which remains in the mammal, said administration in a time
interval prior to or subsequent to or prior to and subsequent to
excision or removal of the tumor.
[0027] This invention discloses a method of prevention of growth of
a tumor from a malignant cell in a host tissue in a mammal
comprising administration to the mammal of a therapeutically effect
amount of a pharmaceutical composition comprising a cell-permeable
fusion protein conjugate comprising a polypeptidic cell-membrane
transport moiety and a Clostridium botulinum C3 exotransferase
unit, or a functional analog thereof, wherein the fusion protein
simultaneously prevents or inhibits at least two of malignant cell
migration, malignant cell proliferation, angiogenesis or tubular
structure formation or capillary network growth proximal to the
malignant cell, and secretion of an active metalloproteinase from
the malignant cell.
[0028] This invention discloses a method of prevention of growth
within a resection margin of a host tissue proximal to a site of
excision or removal of a first tumor of a cancer in a mammal, of a
second tumor comprising a residual tumor cell of the cancer, the
method comprising administration of a therapeutically effective
amount of a pharmaceutical composition comprising a cell-permeable
fusion protein conjugate comprising a polypeptidic cell-membrane
transport moiety and a Clostridium botulinum C3 exotransferase
unit, or a functional analog thereof, said administration being
directly on to the surface of the resection margin or below the
surface of the resection margin or into the tissue proximal to the
resection margin which remains in the mammal, and said
administration being in a time interval prior to, or subsequent to,
or both prior to and subsequent to excision or removal of the first
tumor, wherein the fusion protein simultaneously prevents or
inhibits at least two of residual tumor cell migration, residual
tumor cell proliferation, angiogenesis or tubular structure
formation or capillary network growth proximal to the residual
tumor cell, and secretion of an active metalloproteinase from the
residual tumor cell.
[0029] The invention further provides for the use of the
pharmaceutical composition as defined above for carrying out the
above method or for the manufacture of a medicament for carrying
out the above method.
[0030] In one aspect, the present invention comprises a method of
inhibiting metastases of a systemic cancer into the CNS (central
nervous system) of a mammal comprising administration to the mammal
of a therapeutically effective amount of a pharmaceutical
composition comprising a cell-permeable fusion protein conjugate
comprising a polypeptidic cell-membrane transport moiety and a
Clostridium botulinum C3 exotransferase unit, or a functional
analog thereof, for example a fusion protein such as BA-05.
[0031] In one aspect, a therapeutically effective amount of a
pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, for example a fusion protein such as
BA-05, can exhibit anti-angiogenic activity and is useful in the
treatment of cancer.
[0032] In one aspect, this invention discloses a method of
prevention or inhibition of uncontrolled proliferation and
spreading or migration of a metastatic neoplastic cell of a cancer
in a mammal, comprising administration to the mammal of a
therapeutically effective amount of a pharmaceutical composition
comprising a cell-permeable fusion protein conjugate comprising a
polypeptidic cell-membrane transport moiety and a Clostridium
botulinum C3 exotransferase unit, or a functional analog
thereof.
[0033] In a second aspect, this invention discloses a method of
prevention or inhibition of uncontrolled proliferation and
spreading or migration, within a resection margin of a host tissue
proximal to the site of excision of a tumor of a cancer in a
mammal, of a metastatic neoplastic cell residing in the resection
margin, comprising administration of a therapeutically effective
amount of a pharmaceutical composition comprising a cell-permeable
fusion protein conjugate comprising a polypeptidic cell-membrane
transport moiety and a Clostridium botulinum C3 exotransferase
unit, or a functional analog thereof, said administration being
directly on to the surface of the resection margin or below the
surface of the resection margin or into the tissue proximal to the
resection margin which remains in the mammal, said administration
in a time interval prior to or subsequent to or prior to and
subsequent to excision or removal of the tumor.
[0034] In a third aspect, this invention discloses a method of
prevention of growth of a tumor from a malignant cell in a host
tissue in a mammal comprising administration to the mammal of a
therapeutically effect amount of a pharmaceutical composition
comprising a cell-permeable fusion protein conjugate comprising a
polypeptidic cell-membrane transport moiety and a Clostridium
botulinum C3 exotransferase unit, or a functional analog thereof,
wherein the fusion protein simultaneously prevents or inhibits at
least two of malignant cell migration, malignant cell
proliferation, angiogenesis or tubular structure formation or
capillary network growth proximal to the malignant cell, and
secretion of an active metalloproteinase from the malignant
cell.
[0035] In a fourth aspect, this invention discloses a method of
prevention of growth within a resection margin of a host tissue
proximal to a site of excision or removal of a first tumor of a
cancer in a mammal, of a second tumor comprising a residual tumor
cell of the cancer, the method comprising administration of a
therapeutically effective amount of a pharmaceutical composition
comprising a cell-permeable fusion protein conjugate comprising a
polypeptidic cell-membrane transport moiety and a Clostridium
botulinum C3 exotransferase unit, or a functional analog thereof,
said administration being directly on to the surface of the
resection margin or below the surface of the resection margin or
into the tissue proximal to the resection margin which remains in
the mammal, and said administration being in a time interval prior
to, or subsequent to, or both prior to and subsequent to excision
or removal of the first tumor, wherein the fusion protein
simultaneously prevents or inhibits at least two of residual tumor
cell migration, residual tumor cell proliferation, angiogenesis or
tubular structure formation or capillary network growth proximal to
the residual tumor cell, and secretion of an active
metalloproteinase from the residual tumor cell.
[0036] In a fifth aspect, this invention discloses a use of a
pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, in the manufacture of a medicine for the
prevention or inhibition of uncontrolled proliferation and
spreading or migration of a metastatic neoplastic cell of a cancer
in a mammal.
[0037] In a sixth aspect, this invention discloses a use of a
pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, in the manufacture of a medicine for the
prevention or inhibition of uncontrolled proliferation and
spreading or migration, within a resection margin of a host tissue
proximal to the site of excision of a tumor of a cancer in a
mammal, of a metastatic neoplastic cell residing in the resection
margin, suitable for administration directly on to the surface of
the resection margin or below the surface of the resection margin
or into the tissue proximal to the resection margin which remains
in the mammal, in a time interval prior to or subsequent to or
prior to and subsequent to excision or removal of the tumor.
[0038] In a seventh aspect, this invention discloses a use of a
pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, in the manufacture of a medicine for
prevention of growth of a tumor from a malignant cell in a host
tissue in a mammal, wherein the fusion protein simultaneously
prevents or inhibits at least two of malignant cell migration,
malignant cell proliferation, angiogenesis or tubular structure
formation or capillary network growth proximal to the malignant
cell, and secretion of an active metalloproteinase from the
malignant cell.
[0039] In an eighth aspect, this invention discloses a use of a
pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, in the manufacture of a medicine for
prevention of growth within a resection margin of a host tissue
proximal to a site of excision or removal of a first tumor of a
cancer in a mammal, of a second tumor comprising a residual tumor
cell of the cancer, by administration directly on to the surface of
the resection margin or below the surface of the resection margin
or into the tissue proximal to the resection margin which remains
in the mammal in a time interval prior to, or subsequent to, or
both prior to and subsequent to excision or removal of the first
tumor, wherein the fusion protein simultaneously prevents or
inhibits at least two of residual tumor cell migration, residual
tumor cell proliferation, angiogenesis or tubular structure
formation or capillary network growth proximal to the residual
tumor cell, and secretion of an active metalloproteinase from the
residual tumor cell.
[0040] In a ninth aspect, this invention discloses another aspect
of the previous aspects, wherein the fusion protein conjugate is
BA-05.
[0041] In a tenth aspect, this invention discloses another aspect
of the previous aspects, wherein the cancer is selected from the
group consisting of breast, brain, colon, skin, kidney, and hepatic
cancer.
[0042] In an eleventh aspect, this invention discloses another
aspect of the previous aspects, wherein the cancer is a brain tumor
selected from the group consisting of glial tumors, neuron tumors,
pineal gland tumors, menigeal tumors, tumors of nerve sheath,
lymphomas, malformative tumors, and metastatic tumors located in
the brain derived from tumors of the lung, breast, melanoma,
kidney, and gastrointestinal tract.
[0043] In a twelfth aspect, this invention discloses another aspect
of the previous aspects, wherein the cancer is a brain tumor
selected from the group consisting of anaplastic astrocytoma,
glioblastoma multiform, pilocytic astrocytoma, oligodendroglioma,
ependymoma, myxopapillary ependymoma, subependymoma, choroid plexus
papilloma, neuroblastoma, ganglioneuroblastoma, ganglioneuroma, and
medulloblastoma, pineoblastoma and pineocytoma, meningioma,
meningeal hemangiopericytoma, meningeal sarcoma, Schwannoma
(neurolemmoma) and neurofibroma, Hodgkin's lymphoma, non-Hodgkin's
lymphoma, primary and secondary subtypes of Hodgkin's lymphoma,
primary and secondary subtypes of non-Hodgkin's lymphoma,
craniopharyngioma, epidermoid cysts, dermoid cysts and colloid
cysts.
[0044] In a thirteenth aspect, this invention discloses another
aspect of the previous aspects, wherein the therapeutically
effective amount is about 0.001 micrograms per cc to about 50
micrograms per cc of tissue.
[0045] In a fourteenth aspect, this invention discloses another
aspect of the previous aspects, wherein the therapeutically
effective amount is about 0.0001 micrograms of fusion protein per
cubic centimeter (cc) of tissue to about 100 micrograms per cubic
centimeter of tissue.
[0046] In a fifteenth aspect, this invention discloses another
aspect of the previous aspects, wherein the therapeutically
effective amount is about 1 micrograms per milliliter to about 10
micrograms per milliliter to about 50 micrograms per
milliliter.
[0047] In a sixteenth aspect, this invention discloses another
aspect of the previous aspects, wherein the administration is by
injection, by topical application, or by implantation.
[0048] In a seventeenth aspect, this invention discloses another
aspect of the previous aspects, wherein the administration is
selected from the group consisting of intrarticular, intraocular,
intranasal, intraneural, intradermal, intraosteal, sublingual,
oral, topical, intravesical, intrathecal, intravenous,
intraperitoneal, intracranial, intramuscular, subcutaneous,
inhalation, atomization and inhalation, application directly into a
tumor, application directly into a disease site, application
directly on or into the margins remaining after resection of a
tumor, enteral, enteral together with a gastroscopic procedure, and
ECRP.
[0049] In an eighteenth aspect, this invention discloses another
aspect of the previous aspects, wherein the polypeptidic
cell-membrane transport moiety comprises a peptide containing from
about 5 to about 50 amino acids.
[0050] In a nineteenth aspect, this invention discloses another
aspect of the previous aspects, wherein the Clostridium botulinum
Ce exotransferase unit comprises the amino acid sequence designated
by the sequence of fusion protein BA-05.
[0051] In a twentieth aspect, this invention discloses another
aspect of the previous aspects, wherein the functional analog
comprises a protein exhibiting activity in the range of 50% to 500%
of that of wild type Clostridium botulinum Ce exotransferase.
[0052] In a twenty-first aspect, this invention discloses another
aspect of the previous aspects, wherein the pharmaceutical
composition comprises a pharmaceutically acceptable carrier.
[0053] In a twenty-second aspect, this invention discloses another
aspect of the previous aspects, wherein the pharmaceutical
composition comprises a pharmaceutically acceptable carrier
selected from the group consisting of poly(ethylene-co-vinyl
acetate), PVA, partially hydrolyzed poly(ethylene-co-vinyl
acetate), poly(ethylene-co-vinyl acetate-co-vinyl alcohol), a
cross-linked poly(ethylene-co-vinyl acetate), a cross-linked
partially hydrolyzed poly(ethylene-co-vinyl acetate), a
cross-linked poly(ethylene-co-vinyl acetate-co-vinyl alcohol),
poly-D,L-lactic acid, poly-L-lactic acid, polyglycolic acid, PGA,
copolymers of lactic acid and glycolic acid, polycaprolactone,
polyvalerolactone, poly (anhydrides), copolymers of
polycaprolactone with polyethylene glycol, copolymers of polylactic
acid with polyethylene glycol, polyethylene glycol; and
combinations and blends thereof.
[0054] In a twenty-third aspect, this invention discloses another
aspect of the previous aspects, wherein the pharmaceutical
composition comprises a pharmaceutically acceptable carrier
comprising an aqueous gelatin, an aqueous protein, a polymeric
carrier, a cross-linking agent, and a combination thereof.
[0055] In a twenty-fourth aspect, this invention discloses another
aspect of the previous aspects, wherein the pharmaceutical
composition comprises a pharmaceutically acceptable carrier
comprising a matrix.
[0056] In a twenty-fifth aspect, this invention discloses another
aspect of the previous aspects, wherein the pharmaceutical
composition comprises a pharmaceutically acceptable carrier
comprising water, a pharmaceutically acceptable buffer salt, a
pharmaceutically acceptable buffer solution a pharmaceutically
acceptable antioxidant, ascorbic acid, one or more low molecular
weight pharmaceutically acceptable polypeptide, a peptide
comprising about 2 to about 10 amino acid residues, one or more
pharmaceutically acceptable protein, one or more pharmaceutically
acceptable amino acid, an essential-to-human amino acid, one or
more pharmaceutically acceptable carbohydrate, one or more
pharmaceutically acceptable carbohydrate-derived material, a
non-reducing sugar, glucose, sucrose, sorbitol, trehalose,
mannitol, maltodextrin, dextrins, cyclodextrin, a pharmaceutically
acceptable chelating agent, EDTA, DTPA, a chelating agent for a
divalent metal ion, a chelating agent for a trivalent metal ion,
glutathione, pharmaceutically acceptable nonspecific serum albumin,
and combinations thereof.
[0057] In a twenty-sixth aspect, this invention discloses another
aspect of the previous aspects, wherein the pharmaceutical
composition is sterile.
[0058] In a twenty-seventh aspect, this invention discloses another
aspect of the previous aspects, wherein the pharmaceutical
composition is sterilizable.
[0059] In a twenty-eighth aspect, this invention discloses another
aspect of the previous aspects, wherein the pharmaceutical
composition is sterilized.
[0060] In a twenty-ninth aspect, this invention discloses another
aspect of the previous aspects, wherein the pharmaceutical
composition is in a vial in a unit dosage amount or in an integral
multiple of a unit dosage amount.
[0061] In a thirtieth aspect, this invention discloses another
aspect of the previous aspects, wherein the pharmaceutical
composition is dried.
[0062] In a thirty-first aspect, this invention discloses another
aspect of the previous aspects, wherein the pharmaceutical
composition comprises a dehydrated matrix.
[0063] In a thirty-secondth aspect, this invention discloses
another aspect of the previous aspects, wherein the pharmaceutical
composition comprises a pharmaceutically acceptable carrier.
[0064] In a thirty-third aspect, this invention discloses another
aspect of the previous aspects, wherein the pharmaceutical
composition comprises a fusion protein in a lyophilized matrix.
Antagonism of Rho and Apoptosis
[0065] Mechanisms to control cell proliferation are dysregulated in
cancer. An increased apoptosis in EL4 Murine T lymphoma cells
occurs after Rho inactivation by recombinant C3 exoenzyme. In
NIH3t3 cells, treatment with the Rho kinase inhibitor Y-27632
significantly inhibited anchorage-independent growth. In one
embodiment, inactivation of Rho can prevent tumour cell
proliferation, and the present invention comprises the reduction or
arrest of cell proliferation, or induction of apoptosis by a
cell-permeable fusion protein conjugate comprising a polypeptidic
cell-membrane transport moiety and a Clostridium botulinum C3
exotransferase unit, or a functional analog thereof, for example a
fusion protein such as BA-07. In another embodiment, the present
invention comprises the reduction or arrest of cell proliferation,
or induction of apoptosis by an effective amount of a
pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, for example a fusion protein such as
BA-07.
Antagonism of Rho and Cell Migration.
[0066] Metastatic cancer cells are highly migratory. Inactivation
of Rho can prevent cell migration in certain cell types. C3
transferase and the Rho kinase inhibitor Y-27632 block cellular
invasion by HT29 human colon cancer cells, In a v-Crk-inducible rat
fibroblast 3Y1 cell line, C3 and Y-27632 inhibited v-Crk, resulting
in decreased cell motility. Decreased apoptosis in RhoB -/- cells
in Rho B +/-or RhoB-/- MEF cells treated with doxorubicin,
radiation or Taxol results from the lack of RhoB protein. In
another embodiment, antagonism of Rho can reduce cell migration and
metastasis, and the present invention comprises the inhibition of
cell migration by a cell-permeable fusion protein conjugate
comprising a polypeptidic cell-membrane transport moiety and a
Clostridium botulinum C3 exotransferase unit, or a functional
analog thereof, for example a fusion protein such as BA-07.
[0067] Another embodiment of the present invention comprises the
inhibition of cell migration by an effective amount of a
pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, for example a fusion protein such as
BA-05.
Antagonism of Rho and Matrix Metalloproteinases (MMPs)
[0068] Invasive tumour cells have the property of being able to
degrade the extracellular matrix that surround them by secreting
proteases that degrade the extracellular matrix. One important
class of proteases that are secreted by tumour cells is the matrix
metalloproteinases (MMPs). These enzymes open up paths in the
matrix through which the cancer cells can invade and spread. Tumour
cells can produce different types of MMPs, and MMP are often made
as pro-enzymes that are cleaved and released upon activation. MMP1
cleaves collagen matrix. MMP-2 may play an important role invasion
of lung cancer cells. MMP-9 has also been implicated in tumour cell
invasion. In another embodiment, the present invention comprises
the inhibition of MMP expression, MMP processing or MMP secretion
from a tumor cell, the inhibition by a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, for example a fusion protein such as
BA-07.
[0069] In another embodiment, the present invention comprises the
inhibition of MMP expression, MMP processing or MMP secretion by an
effective amount of a pharmaceutical composition comprising a
cell-permeable fusion protein conjugate comprising a polypeptidic
cell-membrane transport moiety and a Clostridium botulinum C3
exotransferase unit, or a functional analog thereof, for example a
fusion protein such as BA-05.
BA-05 and BA-07 as Rho Antagonists
[0070] BA-05 and BA-07 are genetically engineered forms of C3
exoenzyme. C3 exoenzyme is a bacteriophage-derived secreted protein
discovered in some strains of Clostridium botulinum that transfers
an ADP-ribose group to an asparagine residue of the small
regulatory GTPases, RhoA, RhoB and RhoC. C3 inactivates Rho because
ADP-ribosylation prevents activation of Rho. Novel modifications
that distinguish BA-05 and BA-07 include a C-terminal transport
peptide that allows efficient entry into the cytoplasm, resulting
in a more potent Rho antagonist. BA-05 and BA-07 differ in silent
mutations in the non-enzymatic region. BA-07 allows expression in a
commercial-scale vector for purification of the protein useful as a
therapeutic drug. In one aspect of this invention, a fusion protein
such as BA-07 can be considered to be a cell permeable disruptor of
protein-protein interactions important in signal transduction.
[0071] The present invention provides BA-05 and BA-07 variants such
as a cell-permeable fusion protein conjugate comprising a
polypeptidic cell-membrane transport moiety whose amino acid
sequence can be varied or shorted or elongated or truncated to
comprise a variant of BA-05 and a Clostridium botulinum C3
exotransferase unit whose amino acid sequence can be varied,
elongated, shorted, or truncated in a variant, or a functional
analog thereof, as anti-neoplastic and anti-metastatic
compositions, as well as methods and devices which utilize such
compositions for the treatment of cancer and other malignant
diseases.
[0072] Within one aspect of the present invention, compositions and
methods are provided to alter BA-07 DNA sequence expressed in a
plasmid to enhance the ability to purify large amounts of BA-07 for
formulation in a pharmaceutically acceptable carrier safe for
therapeutic use.
BA-05 and BA-07 are Fusion Proteins According to this
Invention.
[0073] Included in this invention are variants of BA-05 that retain
a proline-rich transport sequence and enough of the C3 transferase
unit to retain enzymatic activity to ADP ribosylsate Rho.
[0074] In accordance with the present invention a conjugate or
fusion protein comprising a therapeutically active agent is
provided whereby the active agent may be delivered across a cell
wall membrane, the conjugate or fusion protein comprising a
transport subdomain(s) or moiety(ies) in addition to an active
agent moiety(ies). More particularly, in accordance with the
present invention a therapeutically active agent as conjugate or
fusion protein is provided comprising a polypeptidic cell-membrane
transport moiety and a Clostridium botulinum C3 exotransferase unit
as a therapeutically active unit, or a functional analog thereof,
wherein the therapeutically active agent can inhibit tumor cell
migration, promote apoptosis of tumor cells, inhibit angiogenesis,
and inhibit production of metalloproteinases associated with tumor
growth.
[0075] It is an advantage that the compositions and methods of the
present invention provide a significant improvement over previous
drugs designed to arrest tumor spread or metastasis because a
single compound of the invention can act as a combination therapy
to arrest several very different aspects of tumor growth and
spread. It is an advantage of that a composition of the present
invention, such as a composition comprising BA-07, can prevent or
retard or inhibit: tumor cell migration, tumor cell proliferation,
angiogenesis at a tumor site, and the secretion of active
metalloproteinases. It is an advantage of the present invention
that pharmaceutically active compounds can penetrate a cancer cell
without reliance on a receptor-based membrane transport mechanism.
It is an advantage of the present invention that pharmaceutically
active compounds can inactivate members of the Rho family GTPases.
It is an advantage of the present invention that pharmaceutically
active compounds are Rho antagonists.
[0076] This invention discloses a method of prevention or
inhibition of uncontrolled proliferation and spreading or migration
of a metastatic neoplastic cell of a cancer in a mammal, comprising
administration to the mammal of a therapeutically effective amount
of a pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof.
[0077] This invention discloses a method of prevention or
inhibition of uncontrolled proliferation and spreading or
migration, within a resection margin of a host tissue proximal to
the site of excision of a tumor of a cancer in a mammal, of a
metastatic neoplastic cell residing in the resection margin,
comprising administration of a therapeutically effective amount of
a pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, said administration being directly on to
the surface of the resection margin or below the surface of the
resection margin or into the tissue proximal to the resection
margin which remains in the mammal, said administration in a time
interval prior to or subsequent to or prior to and subsequent to
excision or removal of the tumor.
[0078] This invention discloses a method of prevention of growth of
a tumor from a malignant cell in a host tissue in a mammal
comprising administration to the mammal of a therapeutically effect
amount of a pharmaceutical composition comprising a cell-permeable
fusion protein conjugate comprising a polypeptidic cell-membrane
transport moiety and a Clostridium botulinum C3 exotransferase
unit, or a functional analog thereof, wherein the fusion protein
simultaneously prevents or inhibits at least two of malignant cell
migration, malignant cell proliferation, angiogenesis or tubular
structure formation or capillary network growth proximal to the
malignant cell, and secretion of an active metalloproteinase from
the malignant cell.
[0079] This invention discloses a method of prevention of growth
within a resection margin of a host tissue proximal to a site of
excision or removal of a first tumor of a cancer in a mammal, of a
second tumor comprising a residual tumor cell of the cancer, the
method comprising administration of a therapeutically effective
amount of a pharmaceutical composition comprising a cell-permeable
fusion protein conjugate comprising a polypeptidic cell-membrane
transport moiety and a Clostridium botulinum C3 exotransferase
unit, or a functional analog thereof, said administration being
directly on to the surface of the resection margin or below the
surface of the resection margin or into the tissue proximal to the
resection margin which remains in the mammal, and said
administration being in a time interval prior to, or subsequent to,
or both prior to and subsequent to excision or removal of the first
tumor, wherein the fusion protein simultaneously prevents or
inhibits at least two of residual tumor cell migration, residual
tumor cell proliferation, angiogenesis or tubular structure
formation or capillary network growth proximal to the residual
tumor cell, and secretion of an active metalloproteinase from the
residual tumor cell.
[0080] In one aspect, the present invention comprises a method of
inhibiting metastases of a systemic cancer into the CNS (central
nervous system) of a mammal comprising administration to the mammal
of a therapeutically effective amount of a pharmaceutical
composition comprising a cell-permeable fusion protein conjugate
comprising a polypeptidic cell-membrane transport moiety and a
Clostridium botulinum C3 exotransferase unit, or a functional
analog thereof, for example a fusion protein such as BA-07.
[0081] In one aspect, a therapeutically effective amount of a
pharmaceutical composition comprising a cell-permeable fusion
protein conjugate comprising a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof, for example a fusion protein such as
BA-07, can exhibit anti-angiogenic activity and is useful in the
treatment of cancer.
[0082] In accordance with the present invention the active agent
region of a fusion protein useful in this invention comprises an
ADP-ribosyl transferase C3 region, or a functional equivalent
thereof. In accordance with the present invention, a preferred
ADP-ribosyl transferase C3 may be selected from the group
consisting of an ADP-ribosyl transferase derived from Clostridium
botulinum and a recombinant ADP-ribosyl transferase.
[0083] Alternatively, C3 can be derived from other sources such as
C. limoseum or Staphylococcus aureus. C3 purified from these
bacteria have enzymatic activity as of C3 from C. botulinum that is
effective to ADP ribosylate Rho and cause inactivation of Rho.
[0084] In one aspect of the present invention a polypeptidic
cell-membrane transport moiety can comprise a proline-rich
transport domain. Examples of a proline-rich transport moiety or
domain can be found in U.S. patent application Ser. No. 10/118,079,
the entire disclosure of which is herein incorporated by reference
in its entirety. As used herein the term "proline-rich region"
refers to any linear sequence of 10 amino acids linked together by
peptide amide bonds within a molecule comprising a peptide or
protein, wherein at least 3 out of the 10 amino acids in the linear
sequence are proline residues, wherein each proline is covalently
linked in a peptide amide bond at its nitrogen and in another
peptide amide bond at its carboxylic (carbonyl) site.
[0085] A proline-rich region in any 10 amino acid sequence within a
peptide can comprise 2 or more proline residues and 8 or fewer
non-proline amino acids.
[0086] For example, in one aspect, a proline-rich region in peptide
comprising a 10 amino acid sequence within a peptide comprising 10
or more amino acids can comprise 2 proline residues and 8
non-proline amino acid residues distributed in any combination
among the 10 amino acids.
[0087] In another aspect, a proline-rich region in peptide
comprising a 10 amino acid sequence within a peptide comprising 10
or more amino acids can comprise 3 proline residues and 7
non-proline amino acid residues distributed in any combination
among the 10 amino acids.
[0088] In another aspect, a proline-rich region in peptide
comprising a 10 amino acid sequence within a peptide comprising 10
or more amino acids can comprise 4 proline residues and 6
non-proline amino acid residues distributed in any combination
among the 10 amino acids.
[0089] In another aspect, a proline-rich region in peptide
comprising a 10 amino acid sequence within a peptide comprising 10
or more amino acids can comprise 5 proline residues and 5
non-proline amino acid residues distributed in any combination
among the 10 amino acids.
[0090] In another aspect, a proline-rich region in peptide
comprising a 10 amino acid sequence within a peptide comprising 10
or more amino acids can comprise 6 proline residues and 4
non-proline amino acid residues distributed in any combination
among the 10 amino acids.
[0091] In another aspect, a proline-rich region in peptide
comprising a 10 amino acid sequence within a peptide comprising 10
or more amino acids can comprise 7 proline residues and 3
non-proline amino acid residues distributed in any combination
among the 10 amino acids.
[0092] In another aspect, a proline-rich region in peptide
comprising a 10 amino acid sequence within a peptide comprising 10
or more amino acids can comprise 8 proline residues and 2
non-proline amino acid residues distributed in any combination
among the 10 amino acids.
[0093] In another aspect, a proline-rich region in peptide
comprising a 10 amino acid sequence within a peptide comprising 10
or more amino acids can comprise 9 proline residues and 1
non-proline amino acid residue distributed in any combination among
the 10 amino acids.
[0094] In another aspect, a proline-rich region in peptide
comprising a 10 amino acid sequence within a peptide comprising 10
or more amino acids can comprise 10 proline residues.
[0095] In another aspect, a "proline-rich region" refers to an
amino acid sequence region of a protein containing more prolines
than that which is generally observed in naturally occurring
proteins (e.g., proteins encoded by the human genome).
[0096] A "proline-rich region" of a peptide in a composition of the
present invention can function to enhance the rate of transport of
a fusion protein of this invention through a cell membrane.
[0097] A non-proline-rich region of a peptide or protein can
comprise a sequence of 10 amino acids covalently linked by peptide
bonds, which region contains zero or one proline residues.
[0098] A call membrane transport-enhancing peptide of a composition
of this invention can comprise one or more than one proline-rich
region, each of which can be the same or different sequence of
amino acids, and each of which is covalently linked together by a
peptide bond or by the peptide bonds comprising one or more
non-proline-rich amino-acid sequence which may each be the same or
different when the non-proline-rich amino-acid sequence comprises
more than 10 amino acids.
[0099] In another aspect of the invention, a polypeptidic
cell-membrane transport moiety suitable for use in compositions and
methods comprising a fusion protein of this invention can be
prepared, for example, by methods modified and adapted for use in
this invention as disclosed in Rojas (1998) 16: 370-375 relating to
a membrane translocating sequence; in Vives (1997) 272: 16010-16017
relating to a Tat-mediated protein delivery; in Wender et al. 2000,
PNAS 24: 13003-13008 related to polyargine sequences; in Derossi
(1996) 271: 18188-18193 relating to antennapedia; in Canadian
patent document 2,301,157 relating to conjugates containing
homeodomain of antennopedia; and in U.S. Pat. Nos. 5,652,122,
5,670,617, 5,674,980, 5,747,641, and 5,804,604 relating to
conjugates containing amino acids of Tat HIV protein (herein, Tat
HIV protein is sometimes referred to as Tat); the entire disclosure
in each of which is herein incorporated by reference in its
entirety.
[0100] Several receptor-mediated transport strategies have been
used to try and improve function of ADP ribosylases. These
strategies or methods include fusing C2 and C3 sequences (Wilde, et
al. (2001) 276: 9537-9542) and use of receptor-mediated transport
with the diptheria toxin receptor (Aullo, et al. (1993) 12:
921-31). These strategies have not produced dramatically increased
potency of C3 activity, unlike the activity that has been found
with BA-05. Moreover, those strategies require receptor-mediated
transport. This requires that the targeted cells must express a
specific receptor, and must express sufficient quantities of that
receptor to significantly improve transport rates. In the case of
dipthera toxin, not all cells express the appropriate receptor,
limiting its potential use. In contrast to these strategies, a
composition of this invention comprising a polypeptide transport
moiety such as, for example, BA-05 is able to cross a cell plasma
membrane by a receptor-independent mechanism.
[0101] In one aspect of this invention, a preferred composition
comprises a cell-permeable fusion protein conjugate comprising a
proline-rich polypeptidic cell-membrane transport moiety comprising
a proline-rich amino acid sequence added to the C-terminal region
of a Clostridium botulinum C3 exotransferase unit, or a functional
analog thereof, in a fusion protein conjugate. An especially
preferred composition is a fusion protein designated BA-05. Fusion
protein compositions comprising a proline-rich amino acid sequence
added to the N-terminal region of a Clostridium botulinum C3
exotransferase unit, or a functional analog thereof, are sometimes
referred to herein as analogs of BA-05.
[0102] In another aspect of this invention, a preferred composition
comprises a cell-permeable fusion protein conjugate comprising a
proline-rich polypeptidic cell-membrane transport moiety comprising
a proline-rich amino acid sequence added to the N-terminal region
of a Clostridium botulinum C3 exotransferase unit, or a functional
analog thereof, in a fusion protein conjugate. Fusion protein
compositions comprising a proline-rich amino acid sequence added to
the N-terminal region of a Clostridium botulinum C3 exotransferase
unit, or a functional analog thereof, are sometimes referred to
herein as variants of BA-05.
[0103] The BA-05 analogs and BA-07 variants of the present
invention each comprise a polypeptidic cell-membrane transport
moiety and a Clostridium botulinum C3 exotransferase unit, or a
functional analog thereof. Functional analogs of a Clostridium
botulinum C3 exotransferase unit can comprise polypeptides such as
biologically active fragments and altered-amino-acid-sequence
analogs of BA-05, wherein the biological activity of such fragments
and altered-amino-acid-sequence analogs of BA-05 derives from a
mechanism of action essentially similar to that of BA-05. Such
fragments comprise or encompass amino acid sequences having
truncations of one or more amino acids relative to that in BA-05.
Such fragments comprise or encompass amino acid sequences having
truncations (or eliminations) of one or more amino acids relative
to the sequence of amino acids in BA-05, wherein a truncation may
originate from the amino or N-terminus, the carboxy or C-terminus,
or from the interior of the protein sequence. Analogs and variants
of BA-05 of the invention can comprise an insertion or a
substitution of one or more amino acids. Compositions of this
invention comprising fragments, analogs and variants useful in this
invention have the biological property of BA-05 that is capable of
inactivation a Rho GTPase and preferably capable of inactivation of
more than one Rho GTPase.
[0104] In another aspect, compositions and methods of this
invention comprise chimeric polypeptides comprising a BA-05 amino
acid sequence or a truncated sequence, fused to and comprising
heterologous amino acid sequences. Such heterologous sequences
encompass those which, when formed into a chimera with BA-05 retain
one or more biological or immunological properties of BA-05, most
preferably the property of being capable of inactivation a Rho
GTPase and even more preferably capable of inactivation of more
than one Rho GTPase.
[0105] In another embodiment, this invention comprises a host cell
transformed or transfected with nucleic acids encoding BA-05
protein or BA-07 chimeric protein. In one aspect, any host cell
which produces a protein comprising a polypeptide that exhibits at
least one of the biological properties of a BA-05 may be used, most
preferably the property of being capable of inactivation a Rho
GTPase and even more preferably capable of inactivation of more
than one Rho GTPase. Representative examples of host cell types
include bacterial, yeast, plant, insect, and mammalian cells. In
addition, BA-05 protein or BA-05 chimeric protein may be produced
in transgenic animals. Transformed or transfected host cells and
transgenic animals can be obtained using materials and methods that
are routinely available to one skilled in the art of molecular and
cell biology. A host cell may contain a nucleic acid sequence
comprising a full-length gene that encodes for BA-05 protein and
which can also include a leader sequence and a C-terminal membrane
anchor sequence. Alternatively, a host cell may contain a nucleic
acid sequence which lacks one leader sequence or which lacks both
of the leader sequences or which lacks the C-terminal membrane
anchor sequence, or which lacks combinations of these sequences. In
addition, nucleic acid sequences which encode a polypeptide
fragment, a polypeptide variant, or a polypeptide analog, each
capable of retention of the biological activity of BA-05, may also
be resident in such host expression systems.
[0106] A Rho antagonist that is a recombinant protein can be made
according to methods of recombinant protein technology known in the
art. A protein of the present invention may be prepared from a
bacterial cell extract, or through the use of recombinant
techniques. BA-05 and related fusion proteins according to the
invention can be produced by transformation (e.g., by transfection,
by transduction, by infection) of a host cell with all or part of a
BA-05-encoding DNA fragment in a suitable expression vehicle or
vector. Suitable expression vehicles include: plasmids, viral
particles, and phage. For insect cells, baculovirus expression
vectors are suitable. The entire expression vehicle or vector, or a
part thereof, can be integrated into the host cell genome by
methods known in the art. In one aspect, use of an inducible
expression vector is preferred.
[0107] Those skilled in the field of molecular biology will
understand that any of a wide variety of expression systems can be
used to provide the recombinant protein. The precise host cell used
is usually not critical to the invention. For example, the BA-05
fusion protein and fusion proteins comprising functional analogs
and variants and fragments of BA-05 of this invention can be
produced in a prokaryotic host (e.g., E. coli or B. subtilis) or in
a eukaryotic host (e.g., Saccharomyces or Pichia; mammalian cells,
e.g., cells designated in the art as COS, NIH 3T3, CHO, BHK, 293,
or HeLa cells; or insect cells).
[0108] To determine the relative and effective Rho antagonist
activity of the compositions of this invention, a tissue culture
bioassay system can be used. BA-05 at a concentration range of from
about 0.01 to about 10 ug/ml is useful and is not toxic to
cells.
[0109] BA-05 is stable at 37.degree. C. for at least 24 hours. The
stability of BA-05 was tested in tissue culture with the following
experiment. The BA-05 was diluted in tissue culture medium, left in
an incubator at 37.degree. C. for 24 hours, then added to the
bioassay system described herein, using retinal ganglion cells as
the test cell type. These cells were able to extend neurites on
inhibitory substrates when treated with C3 stored for 24 hours at
37 C. A minimum stability of 24 hours is achieved.
[0110] Another method to confirm that a compound is a Rho
antagonist can utilize a radioactive assay to detect enzymatic
activity.
[0111] Another method to detect activity can utilize a fluorescent
assay to detect enzymatic activity. For example, BA-05 has at least
two inherent enzymatic activities, glycohydrolase and ADP-ribosyl
transferase. These enzymatic activities can act in a sequential
manner to mono-ADP-ribosylate and inactivate the GTP-binding
protein RhoA by trapping ADP-ribosylated Rho in a complex with
guanine-nucleotide dissociation inhibitor-1 (GDI-1). In the first
step of the reaction, the glycohydrolase activity hydrolyses the
N-glycosidic bond between nicotinamide and adenine dinucleotide
phosphate-ribose (ADP-ribose) in the nicotinamide adenine
dinucleotide (NAD.sup.+) molecule. The second step, catalysed by
the ADP-ribosyltransferase, results in the formation of
ADP-ribose-RhoA. The enzyme assays can measure the glycohydrolase
activity of a fusion protein of this invention such as BA-05 and
BA-07 by following the formation of ADP-ribose.
[0112] In one aspect, the present invention comprises a
pharmaceutical composition useful for suppressing malignant
transformation and metastasis, the pharmaceutical composition
comprising a pharmaceutically acceptable diluent or carrier and a
therapeutically effective amount of composition of this invention,
preferably a fusion protein of this invention.
[0113] In one embodiment, a composition of this invention can
comprise an active member selected from the group consisting of a
drug delivery construct as described herein, a drug conjugate as
described herein, and a fusion protein as described herein (e.g.
including pharmaceutically acceptable chemical equivalents
thereof).
Formulation of BA-05 and Other Compositions of this Invention
[0114] Compositions and methods of this invention can comprise a
pharmaceutically acceptable carrier and a therapeutically effective
amount of a pharmaceutical composition comprising a cell-permeable
fusion protein conjugate comprising a polypeptidic cell-membrane
transport moiety and a Clostridium botulinum C3 exotransferase
unit, or a functional analog thereof. In one aspect, a wide variety
of polymeric carriers may be utilized in a formulation of this
invention. Representative examples of polymeric carriers include
poly(ethylene-co-vinyl acetate) (PVA) and partially hydrolyzed
poly(ethylene-co-vinyl acetate) as poly(ethylene-co-vinyl
acetate-co-vinyl alcohol), any of which can be optionally
crosslinked up to about 40% cross-linked; poly-D,L-lactic acid
including low molecular weight oligomers and high molecular weight
polymers thereof; poly-L-lactic acid including low molecular weight
oligomers and high molecular weight polymers thereof; polyglycolic
acid (PGA); copolymers of lactic acid and glycolic acid;
polycaprolactone; polyvalerolactone; poly (anhydrides), copolymers
of polycaprolactone with polyethylene glycol; copolymers of
polylactic acid with polyethylene glycol, polyethylene glycol; and
combinations and blends thereof. Copolymers can comprise from about
1% to about 99% by weight of a monomer unit. Blends of a first
polymer and a second polymer can comprise from about 1% to about
99% by weight of the first polymer and from about 99% to about 1%
of the second polymer.
Application of BA-05 to Arrest Tumor Spread
[0115] Compositions of the present invention, such as
anti-neoplastic and anti-metastatic compositions, may be formulated
in a variety of forms. For example, in one embodiment, a
pharmaceutical composition comprising a therapeutically effective
amount of a cell-permeable fusion protein conjugate comprising a
polypeptidic cell-membrane transport moiety and a Clostridium
botulinum C3 exotransferase unit, or a functional analog thereof,
can comprise a microsphere, wherein the fusion protein is blended
with or embibed into a matrix comprising a pharmaceutically
acceptable polymeric carrier, optionally in the presence of water
(from about 0.1% to about 15% in one embodiment; alternatively, the
microsphere suspended in a aqueous medium in another embodiment), a
pharmaceutically acceptable buffer salt, a pharmaceutically
acceptable surface active agent, a pharmaceutically acceptable
carbohydrate, a pharmaceutically acceptable emollient, and the
like.
[0116] In another embodiment, a pharmaceutical composition
comprising a therapeutically effective amount of a cell-permeable
fusion protein conjugate comprising a polypeptidic cell-membrane
transport moiety and a Clostridium botulinum C3 exotransferase
unit, or a functional analog thereof, can comprise a paste, a
cream, an ointment, a suppository, a suspension in a
pharmaceutically acceptable oil, and the like.
[0117] In another embodiment, a pharmaceutical composition
comprising a therapeutically effective amount of a cell-permeable
fusion protein conjugate comprising a polypeptidic cell-membrane
transport moiety and a Clostridium botulinum C3 exotransferase
unit, or a functional analog thereof, can comprise a film, for
example wherein the fusion protein is blended or mixed together
with a pharmaceutically F acceptable carrier such as an aqueous
gelatin or an aqueous protein or a polymeric carrier or a
combination thereof, optionally in the presence of a cross-linking
agent species which can crosslink the carrier, the blend then
coated into a film or laminate, optionally in the present of a film
base or a support or matrix, and dried or dehydrated, optionally by
the addition of heat or by lyophilization. Films can be prepared in
unit dosage forms or in bulk and divided and cut into unit dosage
forms.
[0118] In another embodiment, a pharmaceutical composition
comprising a therapeutically effective amount of a cell-permeable
fusion protein conjugate comprising a polypeptidic cell-membrane
transport moiety and a Clostridium botulinum C3 exotransferase
unit, or a functional analog thereof, can comprise an aerosol or
sprayable or aerosolizable composition such as a suspension or
solution of the fusion protein in a pharmaceutically acceptable
fluid such as an aqueous solution of a buffer, optionally with a
tonicity modifier; in a pharmaceutically acceptable fluid such as a
supercritical or liquefied gas such as carbon dioxide or propane or
a low molecular weight fluorocarbon or fluorohydrocarbon or
bromofluorocarbon or chlorofluorocarbon and the like, each of which
is a gas at 37.degree. C. and ambient pressure, the composition
suitable for use, for example, in inhalation or as an aerosol such
as a spray-on-a-tissue-surface application.
[0119] In another aspect, the compositions of the present invention
may be formulated to contain a fusion protein such as BA-05 and an
additional anti-neoplastic and anti-metastatic factor or agent.
[0120] In another aspect, the compositions of the present invention
may be formulated to contain a variety of additional compounds, in
order to provide the formulated fusion protein formulations with
certain physical properties (e.g., elasticity related to
incorporation of a pharmaceutically acceptable plasticizing agent,
a particular melting point such as about 30.degree. C. such as by
use of a polyethylene glycol, or a specified release rate which may
be related to degree of crosslinking or rate of hydration in a
matrix or to solubilization of a matrix, or to preferential
solublization of one component of a matrix which can leave pores in
the matrix through which a carrier fluid such a water can assist in
transport of the fusion protein out of the matrix and into or onto
a desired site in the body of a mammal.
[0121] Within certain embodiments of the invention, compositions
may be combined in order to achieve a desired effect (e.g., two or
more compositions of microspheres of this invention may be combined
in order to achieve a modified net release rate of a fusion protein
of this invention such as both a quick and a slow or prolonged
release of one or more anti-neoplastic and anti-metastatic
factor).
[0122] Compositions of the present invention such as those
comprising BA-05 may be administered either alone, or in
combination with a pharmaceutically acceptable carrier, and/or
pharmaceutically and physiologically compatible excipients,
diluents, tonicity modifying agents, buffers, and the like.
Preferably, such carriers are acceptably nontoxic to a recipient
when used in combination with the dosages and at the
therapeutically effective concentrations of the fusion protein
employed.
[0123] In one aspect, preparation of a pharmaceutical composition
of this invention comprises combining the therapeutically effective
amount of a fusion protein of this invention with one or more
components of a carrier such as water; a pharmaceutically
acceptable buffer salt or buffer solution; a pharmaceutically
acceptable antioxidant such as ascorbic acid; one or more low
molecular weight pharmaceutically acceptable polypeptide (e.g., a
peptide comprising about 2 to about 10 amino acid residues); one or
more pharmaceutically acceptable protein; one or more
pharmaceutically acceptable amino acid such as an
essential-to-human amino acid; one or more pharmaceutically
acceptable carbohydrate or carbohydrate-derived material such as
glucose, sucrose, sorbitol, trehalose, mannitol, maltodextrin,
dextrins, cyclodextrin, and combinations thereof, in one aspect
such carbohydrate preferably comprising a non-reducing carbohydrate
such as a non-reducing sugar when avoidance of the Maillard
reaction (which takes place when components such as a reducing
sugar and an amino acid or peptide or protein react together) is
desired, or in another aspect such carbohydrate preferably
comprising a reducing carbohydrate such a reducing sugar when a
Maillard reaction is desired; a pharmaceutically acceptable
chelating agent such as EDTA, or DTPA, which is a chelating agent
for a metal ion such a divalent metal ion (e.g., Ca+2, Fe+2 and the
like) or a trivalent metal ion (e.g., Fe+3, Y+3, Ln+3, Eu+3 and
other lanthanides, and the like, and which may optionally comprise
a radionuclide); glutathione; and other stabilizers and excipients
known in the are of formulation of a protein material. Preferred
carriers comprise sterile buffered saline at a pH in the range from
about 6 to about 8, preferably at about pH 7.4, and a sterile
isotonic composition comprising saline mixed with pharmaceutically
acceptable nonspecific serum albumin.
[0124] The pharmaceutical compositions of this invention can be
sterile, sterilizable, and sterilized. A preferred method of
sterilization comprises filtration of a pharmaceutical composition
through a 0.2 micron filter in a sterile environment. The sterile
filtered composition can be filled in a vial, preferably into a
sterile vial, in a unit dosage volume amount or in an integral
multiple of a unit dosage amount (e.g., as 2 unit dosage amount, 3
unit dosage amounts, 4 unit dosage amounts, et cetera), preferably
under an inert atmosphere such as sterile nitrogen or argon, and
the vials sealed with a pharmaceutically acceptable stopper,
optionally with a crimp cap. In another aspect, pharmaceutical
composition is dried by removal of water, for example the aqueous
medium can be removed from each vial by a drying process such as by
lyophilization or evaporation to leave a dried or dehydrated matrix
comprising the fusion protein of this invention, before sealing and
capping of the vial. In another aspect, the carrier can comprise a
sterile or sterilizable hypertonic solution of a pharmaceutically
acceptable matrix-forming material or excipient that is compatible
with the fusion protein, for example, such as a pharmaceutically
acceptable non-reducing carbohydrate, together with a compound or
fusion protein of the invention, which hypertonic solution can be
placed in a vial and dried (e.g., by lyophilization) to provide a
matrix comprising the fusion protein and the matrix-forming
excipient, which can be sealed in the vial with a cap. Prior to
use, sterile water can be added to the vial, for example vial
sterile syringe or cannula, which water will dissolve the matrix to
provide a solution or suspension of the fusion protein. Sufficient
water can be added to provide the reconstituted solution or
suspension as an isotonic solution suitable for injectable or
implantable use.
[0125] Pharmaceutical compositions of this invention may be
prepared to be suitable for administration to a mammal, such as a
patient in need of treatment, by a variety of different routes.
Preferred routes of administration include for example
intrarticular, intraocular, intranasal, intraneural, intradermal,
intraosteal, sublingual, oral, topical, intravesical, intrathecal,
intravenous, intraperitoneal, intracranial, intramuscular,
subcutaneous, inhalation or atomization and inhalation, or
application directly into a tumor or disease site or on or into the
margins remaining after resection of a tumor. Other representative
routes of administration comprise enteral optionally together with
a gastroscopic procedure, and colonoscopy, each of which do can be
outpatient procedures and not require full operating room
procedures and prolonged hospitalization, but may require the
presence of medical personnel.
[0126] The pharmaceutical compositions provided herein may be
placed within containers along with packaging material which
provides instructions regarding the use of such materials.
Generally, such instructions will include a description of the
concentration of the active agent, as well as within certain
embodiments, relative amounts or identities of excipient
ingredients or diluents (e.g., water, saline or PBS). In addition,
it may be necessary to reconstitute the anti-neoplastic and
anti-metastatic composition, or pharmaceutical composition to a
pharmaceutically acceptable solution or suspension by the addition
of water and optionally also with shaking or sonication.
[0127] The pharmaceutical compositions of this invention may be
utilized in a wide variety of surgical procedures. For example,
within one aspect of the present invention a pharmaceutical
composition (in the form of, for example, a solution or suspension
or powder suitable from application in an atomized or aerosol or
spray form, or coated in a film) may be applied by spraying (a
sprayable or aerosol-forming form) or by lamination (of a film)
onto a surface of an area of tissue in a patient in need of
treatment prior to, during, or after a surgical removal of a tumor
such as a first tumor, and optionally an amount of normal tissue
immediately proximal to the tumor, from the area of tissue, which
removal leaving a margin of normal tissue around the excision site
of the tumor (tumor margin) in the area of tissue. In one aspect,
this procedure can prevent or substantially retard or inhibit
metastatic growth of a second tumor in the normal surrounding
tissues after removal of the first tumor in the patient. In another
aspect, this procedure can prevent the spread of disease (e.g.,
cancer) to surrounding tissues. Within other aspects of the present
invention, a pharmaceutical composition of the present invention
(e.g., in the form of a spray or an aerosol) may be delivered via
an endoscopic procedure, wherein the composition is sprayed or
aerosolized inside a patient to provide a coating comprising a
fusion protein of this invention on a tumor and/or tissue
surrounding and proximal to a tumor inside a patient, which tumor
is accessed or visualized by endoscopic means. In another aspect,
coating of a pharmaceutical composition on to a tissue proximal to
a tumor or proximal to the site of excision of a tumor can inhibit
angiogenesis in the region of tissue that is coated by the
pharmaceutical composition.
[0128] Within yet other aspects of the present invention, a
pharmaceutical composition of this invention can be coated onto the
surface of an implantable device such as a surgical mesh, wire,
stent, prosthetic device, and the like, to form a coated device,
the coating comprising a fusion protein of this invention and
optionally a polymeric carrier, which coated device may be
implanted in a tissue or organ in a patient as part of a surgical
treatment, such as a surgical removal of a cancerous or benign
tumor, which pharmaceutical composition can prevent or inhibit or
delay or retard growth of a second tumor proximal to the location
of the device, and in another aspect, can also prevent or inhibit
or delay or retard growth of a second tumor in a tissue or organ
remote from the site of the implanted device. The concentration of
the fusion protein can be from 0.01% to about 20% by weight of the
carrier that forms a coating on the device, and the thickness of
the coating can be from about 20 micrometers to about 1 millimeter.
The coating can be applied by coating means known in the art of
coating devices. For example, a coating comprising a pharmaceutical
composition of this invention can be applied to the surface of a
device by means of a spray or aerosol applicator in which the
pharmaceutical composition as a solution in a liquid or fluid
comprising a solvent or as a suspension in a liquid or fluid, which
liquid or fluid can evaporate during and after application as a
spray or an aerosol, is sprayed or aerosolized onto the surface of
a device. Optionally, the coated composition can comprise reactive
chemical functional groups such as olefins or anhydride groups or
active esters or Michael reaction acceptors such as a carbon-carbon
double bond conjugated to a carbonyl group, which double bond can
react with an amine of a protein or peptide or gelatin such as a
carrier protein, which reactive chemical functional groups can
chemically or photochemically form crosslinks in the carrier, which
can prevent solubilization or limit or modify or control swelling
(as a function of concentration of the reactive functional groups
or the time of exposure to crosslinking conditions such as
ultraviolet or gamma irradiation of the coated device) of the
coated carrier by aqueous fluid in the tissue in which the device
is implanted. Control of swelling can be useful to control the rate
at which the fusion protein of this invention migrates from the
device into the tissue proximal to the device and further into the
body of the patient. A wide variety of crosslinking chemistry known
in the art can be useful in this aspect of the invention as long as
the biological activity of the fusion protein is not negated or
eliminated. If an organic solvent or supercritical fluid or
liquefied gas is used in the coating process, then a
pharmaceutically acceptable carrier can be selected which does not
immediately dissolve in the aqueous medium present in tissue
proximal to the site of implantation but permits permeation of the
fusion protein into the aqueous medium.
[0129] Other methods of coating can be used such as dip coating of
a composition, painting, curtain coating, and lamination of a
pharmaceutical composition of this invention.
[0130] In one embodiment, the surface of a device can be first
coated with a first coating layer or primer layer which is then
subsequently coated with a pharmaceutical composition of this
invention as a second coating layer. The primer layer can be
selected to adhere to the surface of the metal or polymeric device
and to adhere to the carrier of the second coating layer. The
primer layer can also comprise immobilized chemical functional
groups (e.g., which can be attached to a polymer in the primer
layer) and which can form crosslinking bonds with the second layer.
The primer layer can optionally contain relatively mobile molecules
comprising for example two or more reactive functional groups,
which molecules can migrate into the second layer and react with
chemical functional groups therein to form crosslinking molecular
bridges.
[0131] In an other embodiment, a pharmaceutically acceptable third
layer can be overcoated on the second layer, the third layer
optionally void of fusion protein. The third layer can serve to
control or modify the release rate of the fusion protein from the
device, for example by being able to dissolve or swell or increase
its permeability with respect to water or the fusion protein as a
function of time to expose the second layer comprising the
pharmaceutical composition of this invention to aqueous media from
the tissue.
[0132] Within one embodiment of the invention a surgical mesh
device comprising a pharmaceutical composition of the present
invention coated on the surface of a wire or polymer mesh may be
utilized or implanted in a patient such as during an abdominal
cancer resection surgical procedure on the patient (e.g.,
subsequent to colon resection) in order to provide support to the
residual tissue structure. The coated mesh device can release a
therapeutically effective amount of the active component (such as
BA-07) of the pharmaceutical composition sufficient to prevent
reoccurrence of the cancer by prevention of growth of a second
tumor proximal to the site of implantation of the coated device.
The fusion protein can migrate from the device at a rate sufficient
to provide a therapeutically effective concentration range in the
tissue proximal to the device.
[0133] A currently preferred concentration range is about 0.0001
micrograms of fusion protein per cubic centimeter (cc) of tissue to
about 100 micrograms per cubic centimeter of tissue can be useful.
A currently more preferred therapeutically effective concentration
range is about 0.001 micrograms per cc to about 50 micrograms per
cc of tissue.
[0134] In another embodiment a coated mesh device can release a
therapeutically effective amount of the active component (such as
BA-07) of the pharmaceutical composition sufficient to prevent
reoccurrence of the cancer by prevention of growth of a second
tumor remote from the site of implantation of the coated
device.
[0135] Within further aspects of the present invention, methods are
provided for treatment of a patient at the site of residual tissue
left at the margin of excision of a first tumor (a tumor excision
site) comprising administration of a pharmaceutical composition of
this invention to a residual tissue at a resection margin of a
first tumor of a cancer subsequent to excision of the first tumor,
such that recurrence of a second tumor of the cancer and formation
of new blood vessels at the site of residual tissue at the first
tumor margin is inhibited. Within one embodiment of the invention,
a pharmaceutical composition of the invention such as a
pharmaceutical composition comprising BA-07 is administered
directly to the residual tissue at a tumor excision site (e.g.,
applied by swabbing, brushing, painting, spraying, aerosolization,
injection, lavage, soaking, or otherwise coating the resection
margins of the tumor with the pharmaceutical composition.
Alternatively, a pharmaceutical composition of this invention such
as a pharmaceutical composition comprising BA-07 in the form of a
surgical paste, ointment, cream, suspension, gel, and the like can
be applied to the surface of the tissue.
[0136] In a preferred embodiment of the invention, a pharmaceutical
composition of this invention comprising a fusion protein such as
BA-07 is applied to residual tissue at the site of excision of a
tumor of the liver such as after a hepatic resection for
malignancy.
[0137] In another preferred embodiment of the invention, a
pharmaceutical composition of this invention comprising a fusion
protein such as BA-07 is applied after a neurosurgical operation
(e.g., related to removal of a tumor of the brain).
[0138] Within one aspect of the present invention, a pharmaceutical
composition of this invention comprising a fusion protein such as
BA-07 may be administered to a tumor resection margin residual
tissue of a wide variety of tumors, including for example, breast,
colon, brain and hepatic tumors. For example, within one embodiment
of the invention, a pharmaceutical composition of this invention
comprising a fusion protein such as BA-05 may be administered to
the residual tissue proximal to the site of removal of a first
tumor of neurological cancer subsequent to excision of the first
tumor, such that spread of cells of the cancer into the residual
tissue and formation of a second tumor and formation of new blood
vessels in the tissue at the residual margin site of the first
tumor are inhibited.
[0139] The brain is highly functionally localized: i.e., each
specific anatomical region is specialized to carry out a specific
function. The location of a cancer in the brain of a patient (and
brain pathology) can be more important than the type of tissue or
tumor type. A relatively small tumor or lesion in a key area of the
brain can be far more devastating than a much larger lesion in a
relatively less important area of the brain. A lesion on the
surface of the brain may be relatively easy to resect surgically,
while a tumor of comparable size but located deep in the brain may
not be relatively easy to resect surgically because access to the
deep tumor could require disruption of intervening tissue such as
by cutting through many vital structures to reach or access and
remove the deep tumor. In addition, benign tumors in the brain can
be dangerous to a patient. A benign tumor may grow in a key area
and cause significant damage to surrounding brain tissue and
function. Although a benign tumor can be cured by surgical
resection, removal of the tumor from deep tissue may not be
possible. If left unchecked a benign tumor can grow, increase in
volume, and cause increased intracranial pressure If such a
condition is left untreated, vital structures in the brain can be
compressed, and death of the patient can result. The incidence of
CNS (central nervous system) malignancies is about 8 to 16 cases
per 100,000 people. The prognosis of a primary malignancy of the
brain is dismal, with a median survival of less than one year, even
following surgical resection. Brain tumors, especially gliomas, are
predominantly a local disease which can recur within about 2
centimeters of the original focus of disease after surgical
removal.
[0140] Representative examples of brain tumors which may be treated
utilizing the compositions and methods described herein include
glial tumors such as anaplastic astrocytoma, glioblastoma
multiform, pilocytic astrocytoma, oligodendroglioma, ependymoma,
myxopapillary ependymoma, subependymoma, choroid plexus papilloma;
neuron tumors such as neuroblastoma, ganglioneuroblastoma,
ganglioneuroma, and medulloblastoma; pineal gland tumors such as
pineoblastoma and pineocytoma; menigeal tumors such as meningioma,
meningeal hemangiopericytoma, meningeal sarcoma; tumors of nerve
sheath cells such as Schwannoma (neurolemmoma) and neurofibroma;
lymphomas such as Hodgkin's lymphoma and non-Hodgkin's lymphoma,
primary and secondary subtypes of Hodgkin's lymphoma, primary and
secondary subtypes of non-Hodgkin's lymphoma (and including
numerous subtypes of these, both primary and secondary);
malformative tumors such as craniopharyngioma, epidermoid cysts,
dermoid cysts and colloid cysts; and metastatic tumors located in
the brain which can be derived from virtually any tumor, the most
common being derived from tumors of the lung, breast, melanoma,
kidney, and gastrointestinal tract.
[0141] In one embodiment of this invention, the pharmaceutical
compositions of the invention may be applied locally, such as
topically or by topical application, in a unit dosage amount. Such
administration can comprise application of a pharmaceutical
composition to the external portion of the epidermis, topical
administration to tissue exposed to topical administration in the
mouth cavity, and the topical instillation onto exposed tissue in
the eye, ear and nose, such that no more than about 10% and
preferably no more than 1% of the unit dose of a fusion protein of
this invention (such as BA-07) enters the blood stream of a patient
directly.
[0142] In another embodiment of this invention, the pharmaceutical
compositions of the invention may be administered systemically such
as by injection into a blood vessel or lymph vessel, for example by
intravenous injection.
[0143] Additional modes of administration include intraperitoneal,
subcutaneous, intramuscular, rectal (e.g, as a suppository dosage
form), vaginal (e.g., as a pessary), and peroral delivery. Dosage
forms of this invention can act as a depot comprising a fusion
protein of this invention, which fusion protein can migrate into
tissue proximal to the site of the depot.
[0144] Compositions for use in topical administration include,
e.g., liquid or gel preparations preferably suitable for
penetration through the skin such as creams, liniments (e.g.,
applied to the skin by friction), lotions, oils, ointments, pastes,
and drops suitable for delivery to tissue of organs such as the
eye, ear, nose.
[0145] In one embodiment of the invention, the fusion protein can
have molecular weight of from about 240,000 daltons to about
300,000 daltons.
[0146] In another embodiment, the compositions provided herein may
be formed into films with a thickness of between 100 micrometers
and 2 millimeters, or thermologically active compositions which are
liquid at one temperature (e.g., above about 25.degree. C.) and
solid or semi-solid (e.g., below about 25.degree. C.).
[0147] Within another aspect of the present invention, methods are
provided for treating residual tissue remaining at a malignant
tumor excision site, comprising administering a pharmaceutical
composition of this invention comprising a fusion protein such as
BA-05 to the residual resection margins of a tumor in a patient
subsequent to excision of the tumor from the patient, such that the
local recurrence of cancer and the formation of new blood vessels
at the site is inhibited.
[0148] Within another aspect of the present invention, methods are
provided for treating a tumor excision site, comprising
administering a composition comprising BA-05 to the resection
margin of a tumor subsequent to excision, such that the local
recurrence of cancer and the formation of new blood vessels at the
site is inhibited.
[0149] Another aspect of the invention comprises a pharmaceutical
composition of this invention in a kit of parts such as a kit
comprising a container and a pharmaceutical composition of this
invention; a kit comprising a sealed vial and a pharmaceutical
composition of this invention; a kit comprising a sterile syringe
and a pharmaceutical composition of this invention; a kit
comprising a sterile syringe containing a pharmaceutical
composition of this invention; a kit comprising a spray or aerosol
applicator and a pharmaceutical composition of this invention; a
kit comprising a brush applicator and a pharmaceutical composition
of this invention; a kit comprising a cannula and a pharmaceutical
composition of this invention; a kit comprising a powder applicator
and a pharmaceutical composition of this invention (which powder
applicator can be used to administer a pharmaceutical dosage form
of this invention as a powder by sprinkle application of a dried
(e.g., lyophilized) powder in a topical application to a tissue; a
kit comprising a coated implantable device and a pharmaceutical
composition of this invention, wherein administration is by
implantation.
[0150] Pharmaceutical products are provided, comprising for
example, a fusion protein such as BA-05 which disrupts Rho
signaling, in a container; and device such as a syringe or tool or
brush or applicator device (such as a spray or aerosol applicator
device in a second container, to be used for applying the fusion
protein such as BA-05 to the tissue forming the walls of a tumor
cavity after surgical removal of the tumor, or applying to the
skin, for example after removal of a malignant melanoma.
[0151] The pharmaceutical composition, the method and use thereof,
in accordance with the present invention are intended to be applied
to mammal. In some embodiments, the term mammal is intended to
include humans, while in other embodiments, the term mammal is
intended to mean non-human mammal.
[0152] These and other aspects of the present invention will become
evident upon reference to the associated detailed description and
attached figures.
BRIEF DESCRIPTION OF THE FIGURES
[0153] FIG. 1 illustrates the effect of a composition of this
invention comprising a fusion protein, BA-07, on the proliferation
of HEC1B human endometrial adenocarcinoma cells as measured by
tritiated thymidine incorporation. The vehicle (10) is phosphate
buffered saline, and BA-07 is used at concentrations of 1 .mu.g/ml
(11), 10 .mu.g/ml (12) and 50 .mu.g/ml (13). Cancer cell
proliferation is reduced in a dose dependent manner.
[0154] FIG. 2 illustrates the effect of a composition of this
invention comprising a fusion protein, BA-07, on the proliferation
of SK-MEL-1 human melanoma cells as measured by tritiated thymidine
incorporation. The vehicle is phosphate buffered saline, and BA-07
is used at concentrations of 1 .mu.g/ml, 10 .mu.g/ml, and 50
.mu.g/ml. Cancer cell proliferation is reduced in a dose dependent
manner.
[0155] FIG. 3A illustrates tube formation by HUVEC endothelial
cells cultured in a Matrigel.TM. matrix. This assay is a cell
culture assay for antiogenesis. Tube formation can be seen in the
control which does not contain a fusion protein of this invention,
FIG. 3A (box 30).
[0156] FIG. 3B illustrates a reduction in tube formation of HUVEC
endothelial cells cultured in a Matrigel.TM. matrix. Cultures
treated with a composition of this invention comprising a fusion
protein, BA-07, had fewer tubes demonstrating an inhibition of
angiogenesis, as shown in FIG. 3B, box 31.
[0157] FIG. 4 shows the inhibition of growth of TK-10 human renal
carcinoma cells by a composition of this invention comprising a
fusion protein, BA-07, as measured by a sulforhodamine B (SRB)
growth inhibition assay. The fusion protein, BA-07, is used at
concentrations of 0.1 .mu.g/ml, 1 .mu.g/ml, 10 .mu.g/ml, and 100
.mu.g/ml. At all concentrations used, cancer cell proliferation is
reduced. Reduction in cancer cell proliferation is dose dependent.
At a concentration of fusion protein of 100 .mu.g/ml, the
composition of the invention induced cell death of cancer
cells.
[0158] FIG. 5 shows the inhibition of growth of HOP-62 Non-small
cell lung cancer cells by a composition of this invention
comprising a fusion protein, BA-07, as measure by a sulforhodamine
B (SRB) growth inhibition assay. The fusion protein, BA-07, is used
at concentrations of 0.1 .mu.g/ml, 1 .mu.g/ml, 10 .mu.g/ml, and 100
.mu.g/ml. At all concentrations used, cancer cell proliferation is
reduced. Reduction of cancer cell proliferation is dose
dependent.
[0159] FIG. 6 shows the inhibition of growth of SF-286 CNS cancer
cells by a composition of this invention comprising a fusion
protein, BA-07, as measured by a sulforhodamine B (SRB) growth
inhibition assay. The fusion protein, BA-07, is used at
concentrations of 0.1 .mu.g/ml, 1 .mu.g/ml, 10 .mu.g/ml, and 100
.mu.g/ml. At all concentrations used, cancer cell proliferation is
reduced. Reduction of cancer cell proliferation is dose
dependent.
[0160] FIG. 7 shows reduction in levels of activated RhoA after
incubation with 10 micrograms per milliliter of fusion protein at 1
hour, 2 hours, 4 hours, 6 hours, and 24 hours after administration
of a pharmaceutical composition comprising a fusion protein of this
invention and a pharmaceutically acceptable vehicle.
[0161] FIG. 8 shows the inhibition of growth (as % growth versus a
vehicle control as reference) of Caki-1 renal carcinoma cells by a
composition comprising a fusion proteins as BA-07, the % growth
measured with an SRB assay at relative concentrations of fusion
protein of 0.1, 1, 10, and 100.
DETAILED DESCRIPTION
[0162] All references set forth herein which describe in more
detail procedures, devices or compositions relevant to this
invention are incorporated by reference in their entirety.
A Method for Making a Fusion Protein of this Invention Such as
BA-05
[0163] BA-05 is the name given to the protein of this invention
made by ligating a cDNA sequence encoding C3 to a fusogenic 19-mer
peptide. To demonstrate the method for making a fusion protein of
this invention, an example of an antennapedia sequence added to the
C-terminus of the C3 polypeptide can be used.
[0164] The stop codon at the 3' end of the DNA sequence can be
replaced with an EcoR1 site by polymerase chain reaction (PCR)
using the primers 5'GAA TTC TTT AGG ATT GAT AGC TGT GCC 3' (SEQ ID
NO: 1) and 5'GGT GGC GAC CAT CCT CCA AAA 3' (SEQ ID NO: 2). The PCR
product can be sub-cloned into a pSTBlue-1 vector (Novagen, city),
then cloned into a pGEX-4T vector using BamH I and Not I
restriction site. This vector can be called pGEX-4T/C3. An
antennapedia sequence useful to add to the 3' end of C3 in
pGEX-4T/C3 can be created by PCR from the pET-3a vector
(Bloch-Gallego (1993) 120: 485-492; and Derossi (1994) 269:
10444-10450), subcloned into a pSTBlue-1 blunt vector, then cloned
into the pGEX-4T/C3, using the restriction sites EcoR I and Sal I,
creating pGEX-4T/C3APL.
[0165] DNA sequence analysis can be performed on the sequence
producing the best response according to this invention.
[0166] pGEX-4T/C3APL clone (Seq ID NO: 3) is a currently preferred
sequence and provides a protein that is a preferred composition of
this invention.
[0167] An example of a C3-like fusion protein is denoted
pGEX-4T/C3APLT (Seq ID NO: 4).
[0168] Two PCR primers are designed to transfer one series of
recombinant constructs (BA-05) into the pET system: Upper primer:
5' ggatctggttccgcgtcatatgtctagagtcgacctg 3' (Seq ID NO:38) Lower
primer: 5' cgcggatccattaggttctccttcttccacttc 3' (SEQ ID NO:39).
[0169] A BamHI site at the 5' end of SEQ ID NO:39 is ggatccatta;
the TGA is replaced by TAAT (atta, in SEQ ID NO:39).
[0170] A program useful to amplify the product using Pfu polymerase
comprises: 95.degree. C. 5' 1 cycle, then 94.degree. C.
2'.fwdarw.56.degree. C. 2'.fwdarw.70.degree. C. 2' 10 cycles, then
94.degree. C. 2'.fwdarw.70.degree. C. 3' 30 cycles and hold at
4.degree. C. A QIAEXII kit (Qiagen) can be used to purify an
agarose gel slice containing a desired DNA band. The insert and
vector are digested with BamHI and NdeI following the instructions
of the manufacturer, purified using agarose gel electrophoresis and
a QIAEXII kit (Qiagen), and incubated together overnight with T4
DNA ligase following the manufacturer's directions.
[0171] E. coli (DH5alpha, or preferably, XL1-Blue) is transformed
with the ligation mixture. The clones can be checked by small scale
induction and SDS-PAGE and can be assured by immunoblotting of the
crude lysates with anti-C3 antibody. Plasmid DNA is purified, and
can be assessed for purity. DNA sequencing can be performed (e.g.,
by LiCor technology in which the entire strand is sequenced for the
full length of the clone).
[0172] A first construct prepared in this fashion (pET3a-BA-07, SEQ
ID NO:7) matched the theoretical DNA sequence of construct
pGEX/APLT with a slight change in the 5.
[0173] A second construct, pET9a-BA-07, can be prepared by
subcloning the insert from pET3a-BA-07 into the pET9a vector by
cleaving the pET3a construct with BamHI and NdeI (New England
BioLabs, Beverly, Mass.) according to the manufacturers
instructions. pET9a plasmid DNA can be cleaved with the same
enzymes. The insert DNA and the vector DNA can be purified by
agarose gel electrophoresis. The insert can be ligated into the new
vector using T4 DNA ligase (New England BioLabs, Beverly, Mass.).
The ligated DNA can be transformed into DH5alpha cells and DNA can
be prepared using QIAGEN mini and maxi kits. Clones can be
characterized by restriction digestion and DNA sequencing of the
insert in both directions (e.g., BioS&T, Lachine, Quebec). The
construct DNA can be transformed into BL21 (DE3) cells and
BL21(DE3)/pLysS cells.
[0174] pET9a-BA-07 protein expression (SEQ ID NO: 57) is superior
in BL21(DE3) compared to BL21(DE3)/pLysS.
[0175] The proteins of the present invention may be prepared from
bacterial cell extracts, or through the use of recombinant
techniques by transformation, transfection, or infection of a host
cell with all or part of a fusion protein-encoding DNA fragment
such as a BA-05-encoding DNA fragment) with an antennapedia-derived
transport sequence in a suitable expression vehicle.
[0176] One skilled in the field of molecular biology will
understand that any of a wide variety of expression systems can be
used to provide a recombinant protein of this invention. The
precise host cell used is not usually critical to the invention,
but variations in yields are expected from one host cell type to
another.
[0177] A fusion protein can be purified by utilising protein
purification techniques known in the art such as affinity
purification techniques or column chromatography using resins that
separate molecules on the basis of properties such as charge, size
and hydrophobicity. Useful affinity techniques include those
employing an antibody (e.g., GST) specific for the fusion protein
being expressed. Histidine-tagged proteins can be selectively
eluted with imidazole-containing buffers. Alternatively,
recombinant protein can be fused to an immunoglobulin Fc domain.
Such a fusion protein can be readily purified using a protein A
column.
[0178] Any of these techniques can be automated and optimized to
provide superior reproducibility and high throughput by use of
commercially available liquid chromatography equipment specialized
for protein purification. It is envisioned that small molecule,
peptide or other mimetics of the above described antagonists are
also encompassed by the invention.
Bioactivity Evaluations of a Pharmaceutical Composition Comprising
a Fusion Protein of this Invention Such as BA-05
Change in Rho Inactivation
[0179] The ability of BA-05 and BA-07 to inactivate Rho can be
demonstrated using a cell culture assay. In this assay the cancer
cell line is plated on tissue culture under the conditions that are
to be utilized. For example, NG108 cells can be plated and left to
proliferate until semi-confluent. NG108 is a neuroblastoma X glioma
formed by Sendai virus-induced fusion of the mouse neuroblastoma
clone N18TG-2 and the rat glioma clone C6 BU-1. The cells are then
harvested, homogenized, and a Rho pull down assay is performed. The
pull down assay uses a "bait" that binds to active Rho. In our
assay we can use, for example, Rho binding domain (RBD) from
Rhoteckin. Other proteins, such as Rho kinase, can also be used.
The "bait" is linked to a bead so that it can be precipitated from
the homogenate. RBD binds to the GTP-Rho in the homogenate and does
not bind to GDP-Rho. In this way, active Rho in the cell culture
can be assessed quantitatively. The extent that BA-07 inactivates
Rho in a cell line can be demonstrated by treating a sample of
cells of the cell line before performing a pull down assay.
[0180] A pull-down assay can be used to determine the amount of
active Rho in a solid tumour. A tumour sample is homogenized in
buffer, a pull-down assay performed, and the amount of GTP Rho can
be compared with the amount found in a non-cancerous tissue. This
assay to detect active Rho can be used as a diagnostic for tumours
that comprise cells with highly activated levels of Rho and which
can respond according to this invention, for example to BA-07
therapy. Measure of activated Rho can be more sensitive than simply
examining expression levels of Rho.
[0181] An in situ pull down assay can be used to detect GTP-Rho in
histological sections. For this assay, cryosections (each about 16
.mu.m in thickness) of tumour samples are incubated, after post
fixation with 4% PFA, with a bacterial lysate containing the
RBG-GST overnight at 4.degree. C. The sections are then washed 3
times in TBS, blocked in 3% BSA for about 1 hr at room temperature
and incubated with an anti-GST antibody (Cell signalling, New
England Biolabs, Mississauga, Canada) and with cell-type specific
antibodies to identify specific cells, and incubated overnight at
4.degree. C. the sections are washed in TBS and incubated for 2 hr
at room temperature with FITC, Texas Red or Rhodamine conjugated
secondary antibodies to reveal immunoreactivity (Jackson
ImmunoResearch, Mississauga, Canada).
DNA and Protein Sequence Details of BA-05
[0182] With respect to this invention, a useful fusion protein
designated as BA-05 has the following DNA coding sequence here
displayed using conventional G, A, T, and C nomenclature. In
oligonucleotide sequences of this invention, the symbols G, C, A,
and T have their conventional meaning.
TABLE-US-00001 GGATCCTCTA GAGTCGACCT GCAGGCATGC AATGCTTATT
CCATTAATCA 50 (SEQ ID NO: 56) AAAGGCTTAT TCAAATACTT ACCAGGAGTT
TACTAATATT GATCAAGCAA 100 AAGCTTGGGG TAATGCTCAG TATAAAAAGT
ATGGACTAAG CAAATCAGAA 150 AAAGAAGCTA TAGTATCATA TACTAAAAGC
GCTAGTGAAA TAAATGGAAA 200 GCTAAGACAA AATAAGGGAG TTATCAATGG
ATTTCCTTCA AATTTAATAA 250 AACAAGTTGA ACTTTTAGAT AAATCTTTTA
ATAAAATGAA GACCCCTGAA 300 AATATTATGT TATTTAGAGG CGACGACCCT
GCTTATTTAG GAACAGAATT 350 TCAAAACACT CTTCTTAATT CAAATGGTAC
AATTAATAAA ACGGCTTTTG 400 AAAAGGCTAA AGCTAAGTTT TTAAATAAAG
ATAGACTTGA ATATGGATAT 450 ATTAGTACTT CATTAATGAA TGTTTCTCAA
TTTGCAGGAA GACCAATTAT 500 TACAAAATTT AAAGTAGCAA AAGGCTCAAA
GGCAGGATAT ATTGACCCTA 550 TTAGTGCTTT TGCAGGACAA CTTGAAATGT
TGCTTCCTAG ACATAGTACT 600 TATCATATAG ACGATATGAG ATTGTCTTCT
GATGGTAAAC AAATAATAAT 650 TACAGCAACA ATGATGGGCA CAGCTATCAA
TCCTAAAGAA TTCGTGATGA 700 ATCCCGCAAA CGCGCAAGGC AGACATACAC
CCGGTACCAG ACTCTAGAGC 750 TAGAGAAGGA GTTTCACTTC AATCGCTACT TGA
783
[0183] pGEX-4TBA-05 Protein Coding Sequence
TABLE-US-00002 Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr
Ser Ile Asn (SEQ ID NO: 37) 1 5 10 15 Gln Lys Ala Tyr Ser Asn Thr
Tyr Gln Glu Phe Thr Asn Ile Asp Gln 20 25 30 Ala Lys Ala Trp Gly
Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 35 40 45 Ser Glu Lys
Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55 60 Asn
Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser 65 70
75 80 Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys
Met 85 90 95 Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp
Pro Ala Tyr 100 105 110 Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn
Ser Asn Gly Thr Ile 115 120 125 Asn Lys Thr Ala Phe Glu Lys Ala Lys
Ala Lys Phe Leu Asn Lys Asp 130 135 140 Arg Leu Glu Tyr Gly Tyr Ile
Ser Thr Ser Leu Met Asn Val Ser Gln 145 150 155 160 Phe Ala Gly Arg
Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly Ser 165 170 175 Lys Ala
Gly Tyr Ile Asp Pro Ile Ser Ala Phe Ala Gly Gln Leu Glu 180 185 190
Met Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu 195
200 205 Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly
Thr 210 215 220 Ala Ile Asn Pro Lys Glu Phe Val Met Aen Pro Ala Asn
Ala Gln Gly 225 230 235 240 Arg His Thr Pro Gly Thr Arg Leu 245
[0184] Primer 1 Useful to Produce BA-07:
TABLE-US-00003 (SEQ ID NO: 38) ggatctggtt ccgcgtcata tgtctagagt
cgacctg
[0185] Primer 2 Useful to Produce BA-07:
TABLE-US-00004 Cgcggatcca ttagttctcc ttcttccact tc (SEQ ID NO:
39)
[0186] pET9a-BA-07 DNA Coding Sequence
[0187] pET9a-BA-07 Protein Sequence
TABLE-US-00005 Met Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser
Ile Asn Gln (SEQ ID NO: 57) 1 5 10 15 Lys Ala Tyr Ser Asn Thr Tyr
Gln Glu Phe Thr Asn Ile Asp Gln Ala 20 25 30 Lys Ala Trp Gly Asn
Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys Ser 35 40 45 Glu Lys Glu
Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile Asn 50 55 60 Gly
Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser Asn 65 70
75 80 Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met
Lys 85 90 95 Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro
Ala Tyr Leu 100 105 110 Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser
Asn Gly Thr Ile Asn 115 120 125 Lys Thr Ala Phe Glu Lys Ala Lys Ala
Lys Phe Leu Asn Lys Asp Arg 130 135 140 Leu Glu Tyr Gly Tyr Ile Ser
Thr Ser Leu Met Asn Val Ser Gln Phe 145 150 155 160 Ala Gly Arg Pro
Ile Ile Thr Lys Phe Lys Val Ala Lys Gly Ser Lys 165 170 175 Ala Gly
Tyr Ile Asp Pro Ile Ser Ala Phe Ala Gly Gln Leu Glu Met 180 185 190
Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu Ser 195
200 205 Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr
Ala 210 215 220 Ile Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala
Gln Gly Arg 225 230 235 240 His Thr Pro Gly Thr Arg Leu 245
[0188] An amino acid residue comprises the group
--NH--CR.sub.1R.sub.2--CO-- when the amino acid residue is located
internally in a peptide. The residue is formed from the
corresponding amino acid NH.sub.2--CR.sub.1R.sub.2--COOH, wherein
R.sub.1 and R.sub.2 are substituents subtended at the central
carbon of the amino acid to comprise the remainder of the amino
acid, by loss of H.sub.2O to form an amide or a peptide bond with
other amino acids, one at the nitrogen and one at the carboxylic
acid carbonyl. An amino acid residue at the N-terminus of a peptide
comprises the group NH.sub.2--CR.sub.1R.sub.2--CO-- in which the
carbonyl is bonded by a peptide bond with another amino acid
residue in the peptide. An amino acid residue at the C-terminus of
a peptide comprises the group --NH--CR.sub.1R.sub.2--COOH in which
the nitrogen is bonded by a peptide bond with another amino acid
residue in the peptide.
[0189] Amino acid residues that can be present in peptide and
protein sequences of this invention are sometimes referred to as
three letter codes or single letter codes commonly used in the art,
which codes comprise: glycine as Gly or G; alanine as Ala or A;
valine as Val or V; leucine as Leu or L; isoleucine as Ile or I;
methionine as Met or M; phenylalanine as Phe or F; tryptophan Trp
or W; proline as Pro or P; serine as Ser or S; threonine as Thr or
T; cysteine as Cys or C; tyrosine as Tyr or Y; asparagine as Asn or
N; glutamine as Gln or Q; aspartic acid Asp or D; glutamic acid Glu
or E; lysine as Lys or K; arginine as Arg or R; and histidine as
His or H. Other amino acids that are not essential amino acids can
be introduced using methods of peptide synthesis known in the art
or by chemical modification such as by acylation (such as by
reaction of a lysine epsilon amine group with an active ester
comprising a carbonyl group to achieve a bond between the epsilon
amine and the carbonyl group), alkylation, urea formation, urethane
formation, and the like to add to the peptide chain chemical
functional groups containing hydrophobic groups (e.g., C-1 to C-18
alkyl and/or aralkyl, which may be saturated, unsaturated, or
contain carbocylic groups such as a proline amide), to add
positively charged groups such as quaternary ammonium alkyl groups
or basic amino groups that can be protonated at a pH found in a
patient with cancer, or both.
[0190] In peptides and proteins of this invention, relatively
non-polar and hydrophobic amino acid residues can comprise G, A, V,
L, I, M, F, W, and P; relatively polar and hydrophilic amino acid
residues can comprise S, T, C, Y, N, and Q; anionic and hydrophilic
amino acid residues can comprise D and E, wherein in each of D and
E a carboxylic acid functional group can be in deprotonated form as
an anionic carboxylate; cationic and hydrophilic amino acid
residues can comprise K in which the basic epsilon primary amino
group can be in protonated form as a cationic ammonium group, H in
which the imidazole nitrogen can be in protonated form to provide
an imidazolium cationic group, and R which can comprise a
protonated amidate group.
Anti-Metastatic Properties of a Pharmaceutical Composition
Comprising a Fusion Protein of this Invention
[0191] In one aspect, a pharmaceutical composition comprising a
fusion protein of this invention can be administered, for example
by injection or by a topical application such as by a coating
method or other method as described herein to a tissue proximal to
or comprising a first tumor in a mammal in need of treatment and
can inhibit migration of a metastatic tumor cell in the mammal, the
tumor cell originating from a site of the first tumor in the
mammal, to a site in healthy or normal tissue of the mammal which
is functionally related and proximal to the tissue in which the
first tumor resides. For example, a pharmaceutical composition
comprising a fusion protein of this invention can be administered
to a kidney tissue proximal to or comprising a kidney tumor a
mammal and can inhibit migration of a metastatic kidney tumor cell
from the tumor in the kidney to healthy tissue in the same kidney
in which the first tumor resides.
[0192] In another aspect, a pharmaceutical composition comprising a
fusion protein of this invention can be administered, for example
by injection or by coating or other method as described herein to a
tissue proximal to or comprising a first tumor in a mammal in need
of treatment, and can inhibit migration of a metastatic tumor cell
in the mammal, the tumor cell originating from a site at the first
tumor in the mammal, to a site in a healthy or normal tissue or
organ in the mammal that is functionally separate from or remote
from the tissue in which the first tumor resides. For example, a
pharmaceutical composition comprising a fusion protein of this
invention be administered to a tissue in the brain comprising a
brain tumor, and can inhibit migration of a metastatic brain tumor
cell into healthy tissues elsewhere in the body such as liver,
spleen or lung tissue.
[0193] In another aspect, after administration of a pharmaceutical
composition comprising a fusion protein of this invention to a
patient in need of treatment, metastatic migration of a malignant
tumor cell is prevented or inhibited, and can substantially reduce
or completely prevent formation of a secondary tumor and can
prevent the spread of malignant cancer in a patient.
Demonstration that a Fusion Protein of this Invention Such as BA-07
can Reduce Cell Motility
[0194] The therapeutic effectiveness of a pharmaceutical
composition comprising a fusion protein of this invention (such as
BA-05) as an anti-metastatic agent can be demonstrated, for example
quantitatively, by means of an in vitro two-dimensional cell
invasion assay. In one such assay, inhibition of metastatic
migration ability of a malignant cell can be measured through the
use of purchased Boyden chambers. Boyden chambers have 2
compartments, wherein the upper and lower compartments are
separated by a membrane. The extent of cell migration is measured
by plating a total number of cells in the upper compartment, and
counting the fraction of that total number of cells that migrate to
the lower compartment. Growth factors can be added to the lower
compartment to enhance cell migration. This model is useful as a
model of cancer cell migration in vivo in a mammal. To test the
ability of a pharmaceutical composition comprising a fusion protein
of this invention (such as BA-07 in sterile phosphate buffered
saline that is isotonic with blood of a mammal) to block migration
of tumor cells, the composition comprising BA-07 is added at
different concentrations of BA-07 to the cancer cells in the upper
compartment. The fraction of the total number of cells that migrate
to the lower compartment in the presence of fusion protein
composition are counted and compared with controls in which the
fusion protein is at zero concentration. The number of cancer cells
that migrate in a control experiment model such migration in a
cancer patient who is not treated with a composition of this
invention. The number of cancer cells that migrate in the presence
of an aliquot of a composition of this invention model such
migration in a cancer patient who is treated with an aliquot of a
composition of this invention. The difference between the latter
and the control experimental cell migration numbers can be
expressed in percent and can range from 100% (i.e., complete
inhibition of migration of a metastatic cell) to about 5%,
preferably from 100% to about 50%, more preferably from about 100%
to about 75%, and most preferably from about 100% to about 90%. A
0% amount can be observed when a first control vehicle is compared
with a second control vehicle which may be the same as the first
control vehicle. A calculation of this percent is given by solving
the expression={(number of cells migrating in the control minus
number of cells migrating in the presence of fusion protein)
divided by (number of cells migrating in the control)} times
100%.
[0195] The therapeutic effectiveness of a pharmaceutical
composition comprising a fusion protein of this invention (such as
BA-05) as an anti-metastatic agent can be demonstrated at least
qualitatively and in one aspect by means of an in vitro
three-dimensional cell invasion assay. In one such assay,
inhibition of metastatic migration ability of a malignant cell can
be measured by the change in the relative ability of a malignant
cell to migrate through a MATRIGEL.TM. matrix after treatment of
the cell with a pharmaceutically acceptable formulation of this
invention comprising a fusion protein of this invention in a
carrier vehicle relative to the ability of the malignant cell to
migrate through the MATRIGEL.RTM. matrix after treatment with the
carrier vehicle as a reference control, the carrier vehicle
containing no fusion protein. In one aspect, a fusion protein of
this invention can inhibit migration of a metastatic tumor cell in
a tissue matrix model to produce an inhibitory change as a
reduction in rate of migration of the cell or as a reduction in the
distance of migration of the cell in a time period.
[0196] The relative change in the distance of migration of a
malignant cell through a model matrix is equal to the difference in
the distance of migration of a cell in the presence of the fusion
protein plus vehicle and the distance of migration of the cell in
the presence of a control vehicle in the absence of the fusion
protein, the difference divided by the distance of migration of the
control vehicle. The relative changes can be expressed in percent
and can range from 100% (complete inhibition of migration of a
metastatic cell) to about 5%, preferably from 100% to about 50%,
more preferably from about 100% to about 75%, and most preferably
from about 100% to about 90%. A 0% amount can be observed when a
first control vehicle is compared with a second control vehicle
which may be the same as the first control vehicle.
[0197] In one embodiment, comparison of efficacies of two fusion
proteins A and B of this invention, which fusion proteins differ
from each other in their amino acid sequence, such as for example
in their respective membrane penetration enhancing sequence, may
provide different observed percentages of inhibition of migration
of a given tumor cell type causes by A and by B. The relative
differences (either absolute percentage such as 100% by A versus
80% by B, or qualitative differences such as A is better than B) in
inhibition may be the same from tumor type to tumor type or may
change from tumor type to tumor type.
[0198] In one aspect, a fusion protein of this invention can
substantially (100%) inhibit metastatic migration of at least one
type of tumor cell.
[0199] In another aspect, a fusion protein of this invention can
substantially (100%) inhibit metastatic migration of at least two
types of tumor cell.
[0200] A useful assay is based on the observed ability of an
invasive tumor cell to migrate through an artificial basement
membrane (MATRIGEL.TM.). In this assay, the change in the ability
of different cancer cell types, each with a differing ability to
migrate through the MATRIGEL.TM. in the absence of treatment with a
composition of this invention, and hence a differing metastatic
invasiveness are evaluated by exposure to a concentration or dose
range of a fusion protein of this invention from 0.1 .mu.g/ml to
100 .mu.g/ml. A preferred concentration range is about 0.0001
micrograms of fusion protein per cubic centimeter (cc) of tissue to
about 100 micrograms per cubic centimeter of tissue.
[0201] Matrigel.TM. Matrix (BD Biosciences) is a solubulized
basement membrane preparation extracted from EHS mouse sarcoma, a
tumor rich in ECM proteins. Its major components are laminin,
collagen IV, heparan sulfate proteoglycans, and entactin. At room
temperature, BD Matrigel.TM. Matrix polymerizes to produce
biologically active matrix material which can mimic mammalian
cellular basement membrane, wherein cells can behave in vitro in a
manner similar to in vivo conditions. Matrigel.TM. Matrix can
provide a physiologically relevant environment for studies of cell
morphology, biochemical function, migration or invasion, and gene
expression.
Inhibition of Angiogenesis by a Pharmaceutical Composition
Comprising a Fusion Protein of this Invention Such as BA-05, and
its Effect on Capillary-Like or Tubule Structures.
[0202] In one aspect, a pharmaceutical composition comprising a
fusion protein of this invention can be administered, for example
by injection or by coating or other method as described herein to a
tissue proximal to or comprising a first tumor in a mammal in need
of treatment and can inhibit the process of angiogenesis of a
metastatic tumor cell or group of tumor cells in the mammal, the
tumor cell or group of cells originating from a site of the first
tumor in the mammal, to a site in healthy or normal tissue of the
mammal which is functionally related and proximal to the tissue in
which the first tumor resides. For example, a pharmaceutical
composition comprising a fusion protein of this invention can be
administered to a kidney tissue proximal to or comprising a kidney
tumor a mammal and can inhibit the process of angiogenesis of a
metastatic kidney tumor cell from the tumor in the kidney in
healthy tissue in the same kidney in which the first tumor
resides.
[0203] In another aspect, a pharmaceutical composition comprising a
fusion protein of this invention can be administered, for example
by injection or by coating or other method as described herein to a
tissue proximal to or comprising a first tumor in a mammal in need
of treatment, and can inhibit the process of angiogenesis
associated with growth of a metastatic tumor cell in the mammal,
the tumor cell originating from a site at the first tumor in the
mammal, to a site in a healthy or normal tissue or organ in the
mammal that is functionally separate from or remote from the tissue
in which the first tumor resides. For example, a pharmaceutical
composition comprising a fusion protein of this invention be
administered to a tissue in the brain comprising a brain tumor, and
can inhibit angiogenesis of a metastatic brain tumor cell in
healthy tissues elsewhere in the body such as in liver, spleen or
lung tissue.
[0204] In another aspect, after administration of a pharmaceutical
composition comprising a fusion protein of this invention to a
patient in need of treatment, angiogenesis associated with
metastatic formation and growth of a malignant tumor cell can be
prevented or inhibited. Administration of a pharmaceutical
composition comprising a fusion protein of this invention to a
patient in need of treatment, can substantially reduce or
completely prevent angiogenesis associated with the formation of a
secondary tumor and can prevent the spread and rooting of malignant
cancer in a patient.
[0205] Formation of new blood vessels by angiogenesis is important
in growth of a first tumor and subsequent growth of a second tumor
formed from a cell or group of cells of the first tumor by
metastasis. Inhibition of angiogenesis by a pharmaceutical
composition comprising a fusion protein of this invention such as
BA-07 can be evaluated in an in vitro system useful for the study
of angiogenesis in the growth of a tumor, i.e., a system comprising
cultivation of endothelial cells in the presence of an extract of
basement membrane (Matrigel). In the experimental observation
conditions, capillary-like structures or tubules associated with
angiogenesis or blood vessel capillary formation can be viewed
under a microscope. The inhibitory effect of a fusion protein of
this invention such as BA-05 on the progress of angiogenesis or on
the formation of a tubular capillary network or on the disruption
of the process or progress of tumor-associated angiogenesis can be
observed by following the disappearance of tubular structures in a
Matrigel assay.
[0206] In a Matrigel assay, Matrigel (about 12.5 mg/mL) is thawed
at about 4.degree. C. The matrix (about 50 uL) is added to each
well of a 96 well plate and allowed to solidify for about 10 min at
about 37.degree. C. The wells containing solid Matrigel are
incubated for about 30 min with HUVEC cells at a concentration of
about 15,000 cells per well. When the cells are adhered, medium is
removed and replaced by fresh medium supplemented with a fusion
protein of this invention such as BA-05 and incubated at 37.degree.
C. for about 6 to about 8 hours. Control wells are incubated with
medium alone. To analyze the growth, tube formation can be
visualized by microscopy at, for example, about 50.times.
magnification. The relative mean length, Yx, of an
angiogenesis-derived capillary network observed in an evaluation of
a pharmaceutical composition comprising a fusion protein, x, of
this invention can be quantified using Northern Eclipse software
according to the instructions.
[0207] Data from a typical Matrigel assay experiment, for example
relating to the effect of a pharmaceutical composition comprising a
fusion protein designated as BA-05 on length of an
angiogenesis-derived capillary network are summarized in Table 1.
These data show that the network formation was inhibited by
approximately 13% to about 20% under the dose and formulation
conditions used versus the inhibition produced by a control vehicle
wherein zero inhibition provides 100% growth. This effect on
angiogenesis can be enhanced by using higher doses of fusion
protein and by preincubation of the HUVEC cells with BA-05 prior to
addition of the cells to Matrigel.
TABLE-US-00006 TABLE 1 Anti-angiogenesis effect of a pharmaceutical
composition comprising a fusion protein, BA-05, on the mean length
of a capillary network in a Matrigel matrix assay Relative mean
length of a capillary Relative mean length of a network produced in
the presence of a Mean length of a capillary capillary network
produced pharmaceutical composition comprising network associated
with in the presence of a vehicle a fusion protein, BA-05, at a
concen- angiogenesis control tration of 10 micrograms per milliter
Y1 100 86.4 Y2 100 78.2 Y3 100 86.7
Tumor Cell Antiproliferation Activity of Pharmaceutical Composition
Comprising a Fusion Protein of this Invention, Such as BA-07
[0208] Demonstration that a fusion protein of this invention, such
as BA-07, can affect multiple aspects of the phenotypes of
malignant cells can be shown by monitoring tritiated thymidine
incorporation in proliferating and growing cells, wherein tritiated
thymidine added to cell culture medium is taken into the cells and
becomes part of the thymidine triphosphate pool therein which is
used by each cell to synthesize DNA. Tritiated thymidine becomes
covalently incorporated into DNA macromolecules in each of the
cells. In cells that are not growing or in cells that are
undergoing death by apoptosis or by necrosis, tritiated thymidine
is either not taken up into the cell or is released into the cell
medium upon lysis of the cell. Tritiated thymidine incorporation
can be used as an overall measurement of the effect of a fusion
protein of this invention such as BA-07 on cell growth, cell
division, cell stasis, and cell death. Cell lines in which BA-07
induces a decrease in 3H-thymidin comprise: human endometrial
cancer cell line HEC 1B, human colorectal cancer cell line CaCo2,
human melanoma cancer cell line SK-MEL-2, and human CNS cancer cell
line A-172.
[0209] Data in Table 2 illustrate the effects of changes in dosage
amounts of a composition comprising a fusion protein of this
invention, BA-07, administered to each of eight representative
human cancer cell lines on tritiated thymidine incorporation into
the eight human cancer cell lines: HEC 1B, Caco-2, SK-MEL-1,
HT1080, MCF7, SW480, 293S, and A172. The dose of fusion protein
BA-07 administered ranged 50-fold from about 1 micrograms per
milliliter to about 10 micrograms per milliliter to about 50
micrograms per milliliter (ug/mL).
TABLE-US-00007 TABLE 2 Response data of human tumor cell lines with
respect to administration of a fusion protein, BA-07, as measured
by incorporation of tritiated thymidine Dose of BA-07 in micrograms
per milliliter Human 50 10 1 Cancer % growth in the presence of a
fusion protein relative Cell Line to that in the presence of a
vehicle alone as a control HEC 1B 10 13 30 Caco-2 21 17 30 SK-MEL-1
34 30 33
[0210] It was unexpectedly observed that these human tumor cell
lines exhibit reduced cell proliferation in the presence of the
fusion protein. Table 2 shows the percent of growth compared to a
control value of 100%.
[0211] Tumor cell lines can be divided into three separate groups
with respect to tritiated thymidine incorporation A composition of
this invention comprising fusion protein BA-07 exhibits a
pronounced effect on cell proliferation in the HEC 1B cell line,
which is an endometrial carcinoma cell line, with an inhibition of
proliferation related to a 50% inhibitory concentration (IC.sub.50)
of less than 1 ug/mL. In addition to the inhibition, there is a
dose-response effect of increasing inhibition at the higher
concentration of BA-07.
[0212] In Caco 2 and SK-MEL-1 cell lines, shown in Table 2, a
fusion protein exhibits a strong inhibitory effect on cell
proliferation as evidenced by lower level of tritiated thymidine
incorporation into the cells of each cell line.
[0213] Abbreviations used in this disclosure.
[0214] ADP adenine dinucleotide phosphate
[0215] ATCC American Type culture collection
[0216] ADPC3 C3 exotransferase; C3 exoenzyme; C3 transferase
[0217] FBS Fetal bovine serum
[0218] HEPES HEPES buffer
[0219] MMP Matrix metalloproteinase
[0220] NAD nicotinamide adenine dinucleotide
[0221] NCI National Cancer Institute
[0222] PBS phosphate buffered saline
[0223] SRB sulforhodamine B
[0224] TCA trichloroacetic acid
[0225] The invention is further illustrated in various embodiments
and aspects by the following non-limiting examples.
Example 1
General Method Useful to Prepare a Fusion Protein According to this
Invention
[0226] To demonstrate a method useful to prepare a fusion protein
of this invention, an example of an antennapedia sequence added to
the C-terminus of the C3 polypeptide is used. The DNA sequence to
be added to the C-terminus can be any DNA sequence that will result
in addition of at least one amino acid to the C-terminus of C3
polypeptide. The stop codon at the 3' end of the DNA can be
replaced with an EcoR1 site by polymerase chain reaction (PCR)
using the primers 5'GAA TTC TTT AGG ATT GAT AGC TGT GCC 3' (SEQ ID
NO: 1) and 5'GGT GGC GAC CAT CCT CCA AAA 3' (SEQ ID NO: 2). The PCR
product can be sub-cloned into a pSTBlue-1 vector (Novagen,
Madison, Wis.), then cloned into a pGEX-4T (Amersham Biosciences,
Baie d'Urfe, Quebec) vector using BamH I and Not I restriction
site. This vector can be called pGEX-4T/C3 and provides a general
method to prepare a fusion protein of this invention. An
antennapedia sequence useful to add to the 3' end of C3 in
pGEX-4T/C3 can be created by PCR from the pET-3a vector containing
the antennapedia sequence (Bloch-Gallego (1993) 120: 485-492; and
Derossi (1994) 269: 10444-10450), subcloned into a pSTBlue-1 blunt
vector, then cloned into the pGEX-4T/C3, using the restriction
sites EcoR I and Sal I, creating pGEX-4T/BA-14. Manipulations of
the target plasmid sequence, such as employing nucleases present in
plasmid DNA or purchased enzymes that result in new DNA sequences,
exonuclease III digestion or site-directed mutagenesis using two
synthetic oligonucleotides containing the desired DNA sequence
incorporated into the pGEX4T/BA-14, can be used to produce novel
DNA sequences that when expressed in an appropriate system produce
proteins that can be purified by standard methods such as affinity
chromatography or standard chromatography using methods such as ion
exchange to separate by charge, size exclusion chromatography to
separate by size, and other methods of protein purification. The
proteins are tested in assays for ability to permeate cells, and
the proteins are tested in assays for their ability to antagonize
Rho activity. DNA sequence analysis can be performed on the plasmid
sequences that produce responses better than that of C3
exotransferase, each compared as a control. pGEX-4T/BA-14 clone
(Seq ID NO: 3) is a currently preferred sequence and provides a
protein that is a preferred composition of this invention. An
example of a C3-like fusion protein is denoted pGEX-4T/BA-05 (SEQ
ID NO:37).
[0227] The proteins of the present invention may be prepared from
bacterial cell extracts, or through the use of recombinant
techniques by transformation, transfection, or infection of a host
cell with all or part of a fusion protein-encoding DNA fragment
such as a BA-05-encoding DNA fragment) with an antennapedia-derived
transport sequence in a suitable expression vehicle.
Example 2
Preparation of a Fusion Protein, BA-05
[0228] The method of example 1 can be used to prepare a fusion
protein designated BA-05, which fusion protein contains an amino
acid sequence. BA-05 is the name given to the protein made by
ligating a cDNA encoding C3 to a cDNA encoding a fusogenic 19-mer
peptide.
[0229] An example of a C3-like fusion protein is denoted
pGEX-4T/BA-05 (SEQ ID NO:37).
[0230] This C3-like fusion protein is prepared by the method
described to manipulate the antennapedia DNA into the pGEX4T/C3
DNA. Twenty or more C3-like fusion proteins are expressed and are
purified as described by the manufacturer (Amersham BioSciences,
Baie D'Urfe, Quebec). The twenty proteins are examined for ability
to inactivate Rho in an in vitro system. Proteins inactivating Rho
to a greater extent, as measured by increased neurite outgrowth
compared to vehicle control or control glutathione-S-transferase
(GST) protein are subjected to further analysis. The products of
this process can include proteins such as BA-14, a protein
described in the general example, or new fusion proteins produced
by the cloning method, which fusion proteins can have properties
such as molecular weight and activity in Rho inactivation bioassays
different than the fusion protein BA-14 molecule or different from
a control of non-fusion protein C3 protein. New fusion proteins can
contain a C3 amino acid sequence, but will be altered at the
carboxyl terminus due to the method employed.
Example 3
Preparation of a Fusion Protein, BA-07
[0231] The method of example 1 can be used to prepare BA-07, which
contains the following amino acid sequence:
TABLE-US-00008 Met Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser
Ile Asn Gln (SEQ ID NO: 57) 1 5 10 15 Lys Ala Tyr Ser Asn Thr Tyr
Gln Glu Phe Thr Asn Ile Asp Gln Ala 20 25 30 Lys Ala Trp Gly Asn
Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys Ser 35 40 45 Glu Lys Glu
Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile Asn 50 55 60 Gly
Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser Asn 65 70
75 80 Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met
Lys 85 90 95 Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro
Ala Tyr Leu 100 105 110 Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser
Asn Gly Thr Ile Asn 115 120 125 Lys Thr Ala Phe Glu Lys Ala Lys Ala
Lys Phe Leu Asn Lys Asp Arg 130 135 140 Leu Glu Tyr Gly Tyr Ile Ser
Thr Ser Leu Met Asn Val Ser Gln Phe 145 150 155 160 Ala Gly Arg Pro
Ile Ile Thr Lys Phe Lys Val Ala Lys Gly Ser Lys 165 170 175 Ala Gly
Tyr Ile Asp Pro Ile Ser Ala Phe Ala Gly Gln Leu Glu Met 180 185 190
Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu Ser 195
200 205 Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr
Ala 210 215 220 Ile Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala
Gln Gly Arg 225 230 235 240 His Thr Pro Gly Thr Arg Leu 245
[0232] Two PCR primers are designed to transfer one series of
recombinant constructs (BA-05) into the pET-9a vector (Novagen,
Madison, Wis.) to create BA-protein when expressed in an
appropriate expression system: Upper primer: 5'
ggatctggttccgcgtcatatgtctagagtcgacctg 3' (Seq ID NO: 38) Lower
primer: 5' cgcggatccattagttctccttcttccacttc 3' (SEQ ID NO: 39). A
BamHI site at the 5' end of Seq ID NO: 39 is ggatccatta; the TGA is
replaced by TAAT (atta, in SEQ ID NO: 39).
[0233] A program useful to amplify the product using Pfu polymerase
comprises: 95.degree. C. 5' 1 cycle, then 94.degree. C.
2'.RTM.56.degree. C. 2'.RTM.70.degree. C. 2' 10 cycles, then
94.degree. C. 2'.RTM.70.degree. C. 3' 30 cycles and hold at
4.degree. C. A QIAEXII kit (Qiagen) can be used to purify an
agarose gel slice containing a desired DNA band. The insert and
vector are digested with BamHI and NdeI following the instructions
of the manufacturer (New England BioLabs, Beverly, Mass.), purified
using agarose gel electrophoresis and a QIAEXII kit (Qiagen), and
incubated together overnight with T4 DNA ligase following the
manufacturer's directions.
[0234] E. coli (DH5alpha, or preferably, XL1-Blue) is transformed
with the ligation mixture. The clones can be checked by small scale
induction and SDS-PAGE and can be assured by immunoblotting of the
crude lysates with anti-C3 antibody. Plasmid DNA is purified, and
can be assessed for purity. DNA sequencing can be performed (e.g.,
by LiCor technology in which the entire strand is sequenced for the
full length of the clone).
[0235] A first construct prepared in this fashion (pET3a-BA-07, SEQ
ID NO:7) matched the theoretical DNA sequence of construct
pGEX/BA-05 with a slight change in the 5' terminus due to the
cloning strategy.
[0236] A second construct, pET9a-BA-07, can be prepared by
subcloning the insert from pET3a-BA-07 into the pET9a vector by
cleaving the pET3a construct with BamHI and NdeI (New England
BioLabs, Beverly, Mass.) according to the manufacturers
instructions. pET9a plasmid DNA can be cleaved with the same
enzymes. The insert DNA and the vector DNA can be purified by
agarose gel electrophoresis. The insert can be ligated into the new
vector using T4 DNA ligase (New England BioLabs, Beverly, Mass.).
The ligated DNA can be transformed into DH5alpha cells and DNA can
be prepared using QIAGEN mini and maxi kits. Clones can be
characterized by restriction digestion and DNA sequencing of the
insert in both directions (e.g., by BioS&T, Lachine, Quebec).
The construct DNA can be transformed into BL21 (DE3) cells,
BL21(DE3)/pLysS cells (Novagen, Madison, Wis.) or another suitable
expression system.
Example 4
General Method for Tritiated Thymidine Uptake as Measure of Cell
Proliferation and Useful to Demonstrate that Fusion Protein BA-07
Reduces Proliferation of Cancer Cells
.sup.3H-Thymidine Incorporation Assays
Medium and Cell Lines
[0237] Cell lines are tested for mycoplasma and found to be
negative prior to the initiation of the studies. Cell lines are
obtained from ATCC. The line HEC-1B is cultured in E-MEM
supplemented with 10% FBS and 1% HEPES. The line Caco-2 is cultured
in E-MEM supplemented with 20% FBS, 1% HEPES, 1 mM sodium pyruvate
and 0.1 mM of non-essential amino acid. The line SK-MEL-1 is
cultured in McCoy's supplemented with 10% FBS and 1% HEPES.
[0238] Volumes of 100 .mu.l of each 2.times. working solution of
fusion protein, positive and vehicle controls are plated in
triplicate in 96-well microtiter plates containing cells
(4.times.10.sup.3/100 .mu.l), yielding a final volume of 200 .mu.l.
The plates are placed at 37.degree. C. incubator with 100% humidity
and 5% CO.sub.2. After about 54 hours of incubation, a volume of 20
.mu.l of tritiated thymidine (3H-thymidine) (ICN, Montreal,
Canada), containing 1.0 .mu.Ci, is added to each well. The
.sup.3H-thymidine is prepared in RPMI-1640 supplemented with 10%
FBS. The cultures are incubated in the same conditions as stated
above, for a further 18 hours. At the end of the incubation, the
cells are harvested with an automated cell harvester (Tomtec), and
the incorporated Counts per minute (cpm) of .sup.3H-thymidine is
measured with a microplate scintillation counter (TopCount NXT,
Packard).
[0239] Demonstration that a fusion protein of this invention, such
as BA-07, can affect multiple aspects of the phenotypes of
malignant cells can be shown by monitoring tritiated thymidine
incorporation in proliferating and growing cells, wherein tritiated
thymidine added to cell culture medium is taken into the cells and
becomes part of the thymidine triphosphate pool therein which is
used by each cell to synthesize DNA. Tritiated thymidine becomes
covalently incorporated into DNA macromolecules in each of the
cells. In cells that are not growing or in cells that are
undergoing death by apoptosis or by necrosis, tritiated thymidine
is either not taken up into the cell or is released into the cell
medium upon lysis of the cell. Tritiated thymidine incorporation
can be used as an overall measurement of the effect of a fusion
protein of this invention such as BA-07 on cell growth, cell
division, cell stasis, and cell death. Cell lines in which BA-07
induces a decrease in 3H-thymidin comprise: human endometrial
cancer cell line HEC 1B, human colorectal cancer cell line CaCo2,
human melanoma cancer cell line SK-MEL-2, and human CNS cancer cell
line A-172.
[0240] Data in Table 2 illustrate the effects of changes in dosage
amounts of a composition comprising a fusion protein of this
invention, BA-07, administered to each of eight representative
human cancer cell lines on tritiated thymidine incorporation into
the eight human cancer cell lines: HEC 1B, Caco-2, SK-MEL-1,
HT1080, MCF7, SW480, 293S, and A172. The dose of fusion protein
BA-07 administered ranged 50-fold from about 1 micrograms per
milliliter to about 10 micrograms per milliliter to about 50
micrograms per milliliter (ug/mL).
Example 5
General Method for Determination of Inhibition of Angiogenesis
[0241] The formation of new blood vessels is studied in a cell
culture model by growing endothelial cells in the presence of a
matrix of basement membrane (Matigel). Human umbilical vein
endothelial cells (HUVEC) are harvested from stock cultures by
trypinization, and are resuspended in growth medial consisting of
EBM-2 (Clonetics), FBS, hydrocortisone, hFGF, VEGF, R3-IGF-1,
ascorbic acid, hEGF, GA-1000, heparin. Matrigel (12.5 mg/mL) is
thawed at 4.degree. C., and 50 mL of Matrigel is added to each well
of a 96 well plate, and allowed to solidify for 10 min. at
37.degree. C. Cells in growth medium at a concentration of 15,000
cells/well are added to each well, and are allowed to adhere for 6
hours. The fusion protein, BA-07, is added to the well at about 10
mg/ml, and in other wells PBS is added as control. The cultures are
allowed to grow for a further 6 to 8 hours. The growth of tubes can
be visualized by microscopy at a magnification of 50.times., and
the mean length of the capillary network is quantified using
Northern Eclipse software. Treatment of the cells in the Matrigel
assay with fusion protein BA-07 reduces tube formation (see FIG.
3).
Example 6
General Method to Demonstrate the Effect of a Fusion Protein on
Inhibition of Proliferation of Cancer Cells
Sulforhodamine B (SRB) Growth Inhibition Assay
[0242] A sulforhodamine B (SRB, available from Molecular Probes)
protein staining assay for the in vitro measurement of cellular
protein content was developed and subsequently adopted for routine
use in the NCI in vitro antitumor screening (Skehan et al., 1990).
The SRB binds to basic amino acids of cellular protein and
colorimetric evaluation provides an estimate of total protein mass
which is related to cell number. This assay is based on the
assumption that dead cells either lyse and are removed during the
procedure, or otherwise do not contribute to the colorimetric end
point. The SRB assay might overestimate the surviving fraction of
cells.
Protocol for SRB Assay
[0243] These tests are conducted on a NCI 60 cell line panel. Cells
are grown in RMPI-L 640 media supplemented with 5% fetal bovine
serum and L-glutamine according to ATCC recommendations for each
cell line. Cells in logarithmic growth are trypsinized and counted.
Cells are inoculated in a 96-well microplate depending on the
doubling time of individual cell lines in 100 .mu.L of growth
media. The microplates are incubated at 37.degree. C., 5% CO.sub.2
and 100% relative humidity for 24 h to resume exponential growth.
After 24 h, two plates of each cell line are fixed in situ with TCA
to represent a measurement of the cell population for each cell
line at the time of test article addition (T.sub.0). The TCA is
removed and the plates are incubated at room temperature for at
least 24 h to dry.
[0244] A fusion protein of this invention is prepared and stored
frozen as a lyophilized powder. It can be reconstituted with
sterile water to form a pharmaceutical composition at about 4.42
microgram of fusion protein per microliter in 10 mM sodium
phosphate, buffer pH 7.4. For each dose point, serial dilutions of
the stock solution are prepared with complete medium containing 50
.mu.g/mL gentamicin to provide fusion protein at 200 .mu.g/mL, 20
.mu.g/mL, 2 .mu.g/mL, 0.2 .mu.g/mL, and 0.02 .mu.g/mL. Aliquots of
100 .mu.L of those test article dilutions are added to the
appropriate well already containing 100 .mu.L of medium to achieve
the final log dilution series doses for the fusion protein.
[0245] After fusion protein (i.e., drug) addition, the microplates
are incubated for an additional period at 37.degree. C., 5%
CO.sub.2 and 100% relative humidity. The assay is terminated by
fixing the protein in the cells to the bottom of the wells using
trichloroacetic acid (TCA). The plates are dried, and then 100
.mu.L of SRB solution at 0.4% (w/v) in 1% acetic acid is added to
each well. The plates are incubated with the protein-binding stain
for 10 min at room temperature.
[0246] After staining, unbound dye is removed by washing 1% acetic
acid, and the plates are dried. Bound stain is solubilized by
adding 200 .mu.L of 10 mM Trizma base while the plates are gently
mixed. The amount of dye is measured by reading the optical density
with a microplate reader at a wavelength of 515 nm.
[0247] Data is analyzed in an Excel spreadsheet.
T.sub.0=Mean absorbance at the time of fusion protein addition
(time 0) C=Mean absorbance for control (no test article containing
drug) T.sub.i=Mean absorbance for fusion protein article (different
dose points in dilution series)
[0248] A percentage growth is calculated for each of the test
article concentrations:
% Growth = [ ( Ti - To ) ( C - To ) ] .times. 100 ##EQU00001##
for concentrations where T.sub.i>T.sub.0
% Growth inhibition = [ ( Ti - To ) ( To ) ] .times. 100
##EQU00002##
for concentrations where T.sub.i<T.sub.0.
[0249] The % growth inhibition can be used to prepare a chart to
compare the effect at different doses. The percentage growth plots
are plotted, and the points where the dose response curves crossed
the PG values of +50, 0, and -50 are used to calculate the
GI.sub.50, TGI and LC.sub.50. GI.sub.50, or concentration required
to inhibit growth 50% is the relevant parameter for the fusion
protein.
Example 7
Specific Use of SRB Assay to Demonstrate Inhibition of Cell Growth
of Human Cancer Cell Lines
TABLE-US-00009 [0250] TABLE 3 GI50 (concentration for 50%
inhibition of cell growth) following fusion protein treatment
measured by SRB assay Cell line Type of Cancer GI50 (.mu.g/mL)
Caki-1 Renal 0.054 TK-10 Renal 0.52 SF-268 CNS 0.326 HOP-62
Non-SCLC 0.269 NCI-H226 Non-SCLC 48.2 HS 578T Breast 36.6
[0251] One fusion protein of this invention, BA.sub.--0_, has an
effect on 4 of 6 human tumor cell lines tested with
.sup.3H-thymidine and an effect on about 10% of the cell lines of
the NCI screen. In the SRB test, it appears to have cytostatic
properties; growth is inhibited compared to controls but the
overall amount of protein does not decrease compared to the amount
measured at time zero (Tz). These results agree with in vivo data
showing that C3 transferase is not highly toxic to animals. The
observed GI.sub.50 values are in the nanomolar to micromolar range,
given a molecular weight of about 27 kDa for the fusion
protein.
Example 8
Detection of Activated Rho by Pull-Down Assay
[0252] NG108 cells are grown in cell culture in the presence of 5%
fetal bovine serum (FBS), 1% penicillin-streptomycin (P/S). After
the cells settle (3-6 hours at 37.degree. C.), BA-05 is added to
the cultures. To lyse the cells, they are washed with ice cold Tris
buffered saline (TBS) and are lysed in modified RIPA buffer (50 mM
Tris pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS,
500 mM NaCl, 10 mM MgCl.sub.2, 10 .mu.g/ml leupeptin, 10 .mu.g/ml
aprotinin, 1 mM phenylmethyl-sulfonyl fluoride (PMSF)). Cell
lysates are clarified by centrifugation at 13,000 g for 10 minutes
at 4.degree. C. and kept at minus 80.degree. C. (-80.degree.
C.).
[0253] Purification of GST-Rho Binding Domain (GST-RBD) is
performed with the cell lysates, which are thawed and resuspended
in 500 uL of RIPA buffer per 1 million cells. To make the GST-Rho
Binding Domain (GST-RBD), bacteria expressing GST-RBD in a PGEX
vector are grown in L-broth (LB) with 100 .mu.l/ml ampicillin.
Overnight cultures are diluted 1:10 into 3600 ml LB and incubated
in a shaking bacterial incubator at 37.degree. C. for 2 hours.
Isopropyl-.beta.-D-thiogalactopyranoside (0.5 mM) is then added to
the incubating cultures for 2 hours. Bacteria are then collected by
centrifugation at 5,000 g for 15 minutes. The pellets are then
resuspended in 40 ml lysis buffer (50 mM Tris pH 7.5, 1% Triton-X,
150 mM NaCl, 5 mM MgCl.sub.2, 1 mM DTT, 10 .mu.g/ml leupeptin, 10
.mu.g/ml aprotinin, 1 mM PMSF). After sonication, the lysates are
spun at 14,000 rpm for 30 minutes at 4.degree. C.
[0254] Frozen cell culture is homogenized in RIPA buffer (50 mM
Tris pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS,
500 mM NaCl, 10 mM MgCl.sub.2, 10 .mu.g/ml leupeptin, 10 .mu.g/ml
aprotinin, 1 mM PMSF). The homogenates and cell lysates are
clarified by two 10-minute centrifugations at 13,000 g at 4.degree.
C. They are then incubated for 50 minutes at 4.degree. C. with
GST-RBD coupled to glutathion agarose beads (Sigma, Oakville,
Canada). The beads are then washed 4 times and eluted in sample
buffer. GTP-bound Rho and total Rho present in tissue homogenates
are detected by western blot. The proteins are transferred to
nitrocellulose and are probed using a monoclonal RhoA antibody
(Santa Cruz, Santa Cruz, Calif.). Bands are visualized with
peroxidase-linked secondary antibodies (Promega, Madison, Wyo.) and
an HRP based chemiluminescence reaction (Pierce, Rockford, Ill.).
Densitometry analysis is performed to quantitate the signal in each
band.
Example 9
Use of Rho Pull-Down Assay as a Diagnostic to Diagnose or Determine
which Tumours can Best Respond to Protein Fusion Therapy Using
BA-07 as an Example
[0255] Biopsy samples of tumours are taken by surgical removal from
a tissue in a mammal (e.g., a human patient) to leave residual
tissue in the margin of the excised tumor when all of a tumor is
removed. The samples are frozen on dry ice or in liquid nitrogen.
Samples of excised tissue of approximately 5 mm.sup.2 are
homogenized in 500 uL RIPA buffer (50 mM Tris pH 7.2, 1% Triton
X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM
MgCl2, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 1 mM PMSF). The
homogenates are clarified by two 10-minute centrifugations at
13,000 g at 4.degree. C. to provide samples for further analysis.
The samples are then incubated for 50 minutes at 4.degree. C. with
GST-RBD coupled to glutathion agarose beads, prepared as described
in example 8. GTP-bound Rho and total Rho present in the tissue
homogenates are detected by western blot.
[0256] To detect which cells in the biopsy sample have activated
Rho, cryostat sections can be prepared. Bacterial lysates of
RBD-GST are clarified by centrifugation at 14,000 rpm for 30
minutes at 4.degree. C. Activated Rho is detected by incubating the
section with bacterial lysate containing RBD-GST. Rat spinal cord
cryosections (about 16 .mu.m thickness) are incubated, after post
fixation with 4% PFA, with the bacterial lysate overnight at
4.degree. C. The sections are then washed 3 times is TBS, blocked
in 3% BSA for 1 hr at room temperature and incubated with anti-GST
antibody (Cell signalling, New England Biolabs, Mississauga,
Canada) and with cell-type specific antibodies. In the case of a
brain tumour neuron-specific antibody (NeuN) or astrocyte-specific
antibody (GFAP) can be used to detect the cell type with activated
Rho to aid in tumour diagnosis. Sections are washed in TBS and
incubated for 2 hr at room temperature with FITC, Texas Red or
Rhodamine conjugated secondary antibodies (Jackson ImmunoResearch,
Mississauga, Canada).
Example 10
General Method to Detect Reduction in Metalloproteinase (MMP)
Activity
[0257] Metalloproteinase activity is detected by zymography whereby
proteolytic activity of enzymes is separated in polyacrylamide gels
under non-reducing conditions. To detect metalloproteinase activity
the glatinolytic activity in culture media from growth of Caki-1
colon carcinoma cells is detected by gelatin zymography. The Caki-1
cells are incubated with BA-07 at 0.1, 1.0 or 10 .mu.g/ml or buffer
as control for 24 hr. An aliquot (25 .mu.L) of the culture media is
subjected to SDS/PAGE with 7.5% polyacrylamide containing 1 mg/ml
gelatin, and the polypeptides are separated under non-reducing
conditions. To assess MMP activity, SDS is removed by incubation
for 30 min at room temperature in 2.5% (v/v) Triton X-100. This
step is repeated, followed by five rinses with ddH.sub.20, Next,
the gel is incubated for 20 h at 37.degree. C. in a buffer
containing 50 mM Tris-HCl, pH 7.6, 0.2 M NaCl, 5 mM CaCl.sub.2, and
0.02% (v/v) Brij-35. The gel is stained with Coomassie Brilliant
Blue R-250, and destained. Enzyme activity on the gelatin substrate
is detectable as transparent bands in a blue background. The
identity of the MMP enzyme with gelatinase activity is assessed
with a positive control such as, in these experiments, HT-1080.
Example 11
Detection of Reduction of Metalloproteinase Activity after
Treatment with BA-07
[0258] The method of example 10 is employed using the fusion
protein BA-07. A reduction in metalloproteinase activity is
observed.
Example 12
Formulation of a Fusion Protein in a Sterile Solution
[0259] A therapeutically effective unit dosage amount of a fusion
protein of this invention such as BA-07 is dissolved in a unit
dosage volume of a sterile isotonic solution such as sterile
isotonic PBS to form a unit dosage amount of solution, which is
filtered through a 0.2 micron filter under aseptic conditions. The
filtrate is collected into a sterile vial under an inert atmosphere
(e.g., nitrogen or argon). The vial is then sealed with a sterile
septum and crimp cap, and stored at room temperature. The unit
dosage amount of solution in the vial containing the fusion protein
such as fusion protein BA-07 can be administered to a patient by
injection such as by intravenous delivery, infusion, or by
injection directly into a tumor site in a mammal such as tumor in a
human patient, or by injection into the margins of the site of
excision of a tumor in the tissue of a mammal such as a tissue in a
human patient.
[0260] Two or more vials each containing a unit dosage amount can
be prepared in similar fashion, and the unit dosage amounts can be
administered by injection over a therapeutically effective time of
treatment to a patient in need of treatment. In such fashion, a
sequence of unit dosage amount administrations can be made to the
tissue of a patient or systemically to a patient. For example, a
unit dosage amount of a fusion protein composition can be
administered once a day to a patient, or once every two days to a
patient, or once a week to a patient. In addition, a
therapeutically effective unit dosage amount of a pharmaceutical
composition comprising a fusion protein can be administered to a
patient having a tumor in a tissue of the patient systemically on
one or more occasions before the tumor is excised and/or into the
diagnostically identified margins of a tumor in the patient on one
or more occasions before the tumor is excised such as by surgical
excision, and/or directly into the tumor tissue on one or more
occasions before the tumor is excised, and/or systemically on one
or more occasions after the tumor is excised, and/or directly into
the residual margins of the tumor after the tumor is excised. The
number of such repeated unit dosage administrations and the amount
of fusion protein per unit dosage form can vary from patient to
patient and from tumor type to tumor type and tumor size in order
to prevent growth of a second tumor in the presence of a first
tumor or after removal of a first tumor.
Example 13
A Lyophilized Formulation
[0261] A solution comprising unit dosage amount of a composition of
this invention comprising a fusion protein such as BA-07 dissolved
in an pharmaceutically acceptable isotonic aqueous medium
comprising a pharmaceutically acceptable buffer salt and/or a
readily water-soluble pharmaceutically acceptable carbohydrate
(preferably a pharmaceutically acceptable non-reducing sugar or a
cyclodextrin) is sterile-filtered (e.g. through a 0.2 micron
filter) under aseptic conditions, the filtrate is placed in a
sterilized vial, the filtrate is frozen, the frozen aqueous
solution is lyophilized aseptically at reduced pressure in a
pharmaceutically acceptable lyophilizer to leave a dried matrix
comprising the fusion protein in the vial, the vial is returned to
atmospheric pressure under a sterile inert atmosphere, the vial is
sealed with a sterile stopper (e.g. together with a crimp cap). The
sealed vial is labeled with its contents and dosage amount and
placed in a kit together with a second sealed sterile vial which
contains sterilized water for injection in an amount useful to
transfer into the first vial containing the lyophilized fusion
protein in order to reconstitute the fusion protein matrix to a
solution as a unit dosage form. In another embodiment, the fusion
protein can be dissolved in a starting volume of aqueous medium
which comprises a hypertonic aqueous medium, the solution sterile
filtered, the filtrate filled into a vial, and lyophilized to form
a dried matrix. This dried matrix can be dissolved or reconstituted
in a larger-than-original volume of sterile water, the larger
volume sufficient to form an isotonic solution for injection.
Alternatively, a hypertonic solution can be used for administration
by infusion into a drip bag containing a larger volume of isotonic
aqueous medium such that the hypertonic solution is substantially
diluted. Optionally, a vial containing a volume of sterile water in
an amount suitable to reconstitute the matrix to a unit dosage form
is distributed as a kit with the lyophilized protein. Preferably
the reconstituted composition comprises an isotonic solution. The
fusion protein can be used for intravenous delivery, and/or
infusion, and/or direct injection into a tumor with this
formulation in a manner similar to that in the previous
example.
Example 14
Formulation in a Polymer
[0262] A composition of this invention comprising a fusion protein,
such as BA-07, is formulated by blending into a co-polymer of
polyglycolic acid (PGA) and polylactic acid (PLA). PGA/PLA
co-polymers can degrade 2-6 months after implantation, depending on
the ratio of PGA to PLA. In one formulation, PGA/PLA are used and
dissolved in a non-denaturing organic solvent at concentrations of
0.5-50%, preferably 1.0-3.0%. The polymer solution can then be
spread with a drawdown knife or cast on the surface of a
polysaccharide-based film or foam or applied by spray or dip
coating or other useful means, and then dried by removal of
solvent. Composite mesh such as a mesh comprising a
pharmaceutically acceptable dissolvable and/or degradable polymer
can be made to incorporate a fusion protein such as BA-07, which
will be released as the mesh degrades. The mesh can be implanted in
the site of surgical resection of a tumor, and the fusion protein
will be released to prevent metastasis and growth of any remaining
tumor cells.
Example 15
General Method to Treat an Excised Tumor Margin
[0263] A composition of this invention comprising a fusion protein,
such as BA-07, formulated in a pharmaceutically acceptable cream
can be used to treat an excision site from the skin. An example is
the treatment of malignant melanoma, where such a cream is put on
the skin surrounding the excision site of the tumor. In one aspect,
such a formulation of a cream containing the fusion protein such as
BA-07 can be administered to the skin prior to excision of the
tumor and used to treat the tumor between the period of first
biopsy and before positive histological diagnosis. The cream when
applied to the tumor site can prevent the spread and metastasis of
the tumor.
Example 16
Prevention of a Second Tumour Growing in a Tumour Margin
[0264] A composition of this invention comprising a fusion protein,
such as BA-07, for example such as an aqueous solution as described
above or such as formulated in a surgical adhesive gel, such as a
fibrin adhesive or a hydrogel, can be used to treat the area of a
surgical resection of a tumor. An example is the treatment of a
healthy colon after colonectomy for a colon cancer. The healthy
colon tissue that otherwise surrounded the tumor region prior to
excision of the tumor can be treated with a fusion protein
composition such as BA-07, after removal of the tumor and
associated tissue, in a surgical gel such as a fibrin sealant, and
will be useful to prevent formation of additional lesions in the
residual tissue.
Example 17
General Method to Demonstrate Preclinical Efficacy in a Mammal
[0265] A melanoma cell line is implanted subcutaneously in a first
group of nude mice (Charles River Laboratories). Tumors are grown
mice of the first group of mice, harvested, and transplanted
individually into each mouse (one tumor per mouse) of a second
group of mice. A daily injection of a pharmaceutical composition of
this invention comprising an effective dose of a fusion protein
such as BA-07, which is estimated to be in the range of 10-100
ug/mL of tumor volume, in a pharmaceutically acceptable vehicle is
administered to each mouse in the second group of mice. Control
animals are injected with vehicle as a control. Tumor growth is
measured, and histology performed to measure markers from malignant
keratinocytes such as gamma immuno protein 10 (IP10). The
composition comprising the fusion protein prevents or substantially
inhibits the growth of tumors in the second mice.
Example 18
Use of a Composition Comprising a Fusion Protein Applied to the
Surface of an Implanted Breast Device in the Prevention of
Recurrence of Breast Cancer
[0266] A therapeutically effective amount of a pharmaceutical
composition of this invention comprising a fusion protein is coated
onto the surface of a pharmaceutically acceptable breast implant. A
tumor is excised from the tissue of a breast in a patient,
optionally with co-administration (pre and/or post operative) of a
pharmaceutical composition of this invention as described
hereinabove. The void created by the excision of the tumor is
filled at least in part with the breast implant coated with a
pharmaceutical composition comprising a fusion protein, and the
wound created by the excision and/or implantation is closed. Growth
of a second tumor in the residual tumor margin tissue is
substantially inhibited or prevented.
Example 19
General Method for Preparation of a Fusion Protein
DNA Sequence of a Representative Fusion Protein, BA-14
[0267] Nucleotide Sequence of Fusion Protein BA-14 (SEQ ID
NO:3)
TABLE-US-00010 ggatcctcta gagtcgacct gcaggcatgc aatgcttatt
ccattaatca aaaggcttat 60 tcaaatactt accaggagtt tactaatatt
gatcaagcaa aagcttgggg taatgctcag 120 tataaaaagt atggactaag
caaatcagaa aaagaagcta tagtatcata tactaaaagc 180 gctagtgaaa
taaatggaaa gctaagacaa aataagggag ttatcaatgg atttccttca 240
aatttaataa aacaagttga acttttagat aaatctttta ataaaatgaa gacccctgaa
300 aatattatgt tatttagagg cgacgaccct gcttatttag gaacagaatt
tcaaaacact 360 cttcttaatt caaatggtac aattaataaa acggcttttg
aaaaggctaa agctaagttt 420 ttaaataaag atagacttga atatggatat
attagtactt cattaatgaa tgtctctcaa 480 tttgcaggaa gaccaattat
tacacaattt aaagtagcaa aaggctcaaa ggcaggatat 540 attgacccta
ttagtgcttt tcagggacaa cttgaaatgt tgcttcctag acatagtact 600
tatcatatag acgatatgag attgtcttct gatggtaaac aaataataat tacagcaaca
660 atgatgggca cagctatcaa tcctaaagaa ttcgtgatgg aatcccgcaa
acgcgcaagg 720 cagacataca cccggtacca gactctagag ctagagaagg
agtttcactt caatcgctac 780 ttgacccgtc ggcgaaggat cgagatcgcc
cacgccctgt gcctcacgga gcgccagata 840 aagatttggt tccagaatcg
gcgcatgaag tggaagaagg agaactga 888
[0268] Protein Sequence of Fusion Protein BA-14 SEQ ID NO:4):
TABLE-US-00011 Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr
Ser Ile Asn 1 5 10 15 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe
Thr Asn Ile Asp Gln 20 25 30 Ala Lys Ala Trp Gly Asn Ala Gln Tyr
Lys Lys Tyr Gly Leu Ser Lys 35 40 45 Ser Glu Lys Glu Ala Ile Val
Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55 60 Asn Gly Lys Leu Arg
Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser 65 70 75 80 Asn Leu Ile
Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95 Lys
Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105
110 Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile
115 120 125 Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn
Lys Asp 130 135 140 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met
Asn Val Ser Gln 145 150 155 160 Phe Ala Gly Arg Pro Ile Ile Thr Gln
Phe Lys Val Ala Lys Gly Ser 165 170 175 Lys Ala Gly Tyr Ile Asp Pro
Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185 190 Met Leu Leu Pro Arg
His Ser Thr Tyr His Ile Asp Asp Met Arg Leu 195 200 205 Ser Ser Asp
Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220 Ala
Ile Asn Pro Lys Glu Phe Val Met Glu Ser Arg Lys Arg Ala Arg 225 230
235 240 Gln Thr Tyr Thr Arg Tyr Gln Thr Leu Glu Leu Glu Lys Glu Phe
His 245 250 255 Phe Asn Arg Tyr Leu Thr Arg Arg Arg Arg Ile Glu Ile
Ala His Ala 260 265 270 Leu Cys Leu Thr Glu Arg Gln Ile Lys Ile Trp
Phe Gln Asn Arg Arg 275 280 285 Met Lys Trp Lys Lys Glu Asn 290
295
[0269] To demonstrate the method for making a fusion protein of
this invention, an example of an antennapedia sequence added to the
C-terminus of the C3 polypeptide is useful. A DNA sequence to be
added to the C-terminus can be any DNA sequence that will result in
addition of at least one amino acid to the C-terminus of a peptide
comprising a C3 polypeptide.
[0270] First, pGEX2T-C3 plasmid DNA (N. Lamarche, McGill
University) is prepared using standard methods. The stop codon at
the 3' end of the DNA can be replaced with an EcoR1 site by
polymerase chain reaction (PCR) using the primers 5'GAA TTC TTT AGG
ATT GAT AGC TGT GCC 3' (SEQ ID NO: 1) and 5'GGT GGC GAC CAT CCT CCA
AAA 3' (SEQ ID NO: 2). The PCR product can be sub-cloned into a
pSTBlue-1 vector (Novagen, Madison, Wis.), then cloned into a
pGEX-4T (Amersham Biosciences, Baie d'Urfe, Quebec) vector using
BamH I and Not I restriction site. This vector can be called
pGEX-4T/C3 and provides a general method to prepare a fusion
protein of this invention. An antennapedia sequence useful to add
to the 3' end of C3 in pGEX-4T/C3 can be created by PCR from the
pET-3a vector containing the antennapedia sequence (Bloch-Gallego
(1993) 120: 485-492; and Derossi (1994) 269: 10444-10450),
subcloned into a pSTBlue-1 blunt vector, then cloned into the
pGEX-4T/C3, using the restriction sites EcoR I and Sal I, creating
pGEX-4T/BA-14.
[0271] Nucleotide Sequence of BA-14 (SEQ ID NO: 3)
TABLE-US-00012 ggatcctcta gagtcgacct gcaggcatgc aatgcttatt
ccattaatca aaaggcttat 60 tcaaatactt accaggagtt tactaatatt
gatcaagcaa aagcttgggg taatgctcag 120 tataaaaagt atggactaag
caaatcagaa aaagaagcta tagtatcata tactaaaagc 180 gctagtgaaa
taaatggaaa gctaagacaa aataagggag ttatcaatgg atttccttca 240
aatttaataa aacaagttga acttttagat aaatctttta ataaaatgaa gacccctgaa
300 aatattatgt tatttagagg cgacgaccct gcttatttag gaacagaatt
tcaaaacact 360 cttcttaatt caaatggtac aattaataaa acggcttttg
aaaaggctaa agctaagttt 420 ttaaataaag atagacttga atatggatat
attagtactt cattaatgaa tgtctctcaa 480 tttgcaggaa gaccaattat
tacacaattt aaagtagcaa aaggctcaaa ggcaggatat 540 attgacccta
ttagtgcttt tcagggacaa cttgaaatgt tgcttcctag acatagtact 600
tatcatatag acgatatgag attgtcttct gatggtaaac aaataataat tacagcaaca
660 atgatgggca cagctatcaa tcctaaagaa ttcgtgatgg aatcccgcaa
acgcgcaagg 720 cagacataca cccggtacca gactctagag ctagagaagg
agtttcactt caatcgctac 780 ttgacccgtc ggcgaaggat cgagatcgcc
cacgccctgt gcctcacgga gcgccagata 840 aagatttggt tccagaatcg
gcgcatgaag tggaagaagg agaactga 888
[0272] Protein Sequence of Fusion Protein BA-14 (SEQ ID NO:4):
TABLE-US-00013 Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr
Ser Ile Asn 1 5 10 15 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe
Thr Asn Ile Asp Gln 20 25 30 Ala Lys Ala Trp Gly Asn Ala Gln Tyr
Lys Lys Tyr Gly Leu Ser Lys 35 40 45 Ser Glu Lys Glu Ala Ile Val
Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55 60 Asn Gly Lys Leu Arg
Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser 65 70 75 80 Asn Leu Ile
Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95 Lys
Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105
110 Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile
115 120 125 Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn
Lys Asp 130 135 140 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met
Asn Val Ser Gln 145 150 155 160 Phe Ala Gly Arg Pro Ile Ile Thr Gln
Phe Lys Val Ala Lys Gly Ser 165 170 175 Lys Ala Gly Tyr Ile Asp Pro
Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185 190 Met Leu Leu Pro Arg
His Ser Thr Tyr His Ile Asp Asp Met Arg Leu 195 200 205 Ser Ser Asp
Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220 Ala
Ile Asn Pro Lys Glu Phe Val Met Glu Ser Arg Lys Arg Ala Arg 225 230
235 240 Gln Thr Tyr Thr Arg Tyr Gln Thr Leu Glu Leu Glu Lys Glu Phe
His 245 250 255 Phe Asn Arg Tyr Leu Thr Arg Arg Arg Arg Ile Glu Ile
Ala His Ala 260 265 270 Leu Cys Leu Thr Glu Arg Gln Ile Lys Ile Trp
Phe Gln Asn Arg Arg 275 280 285 Met Lys Trp Lys Lys Glu Asn 290
295
[0273] The fusion proteins of the present invention may be prepared
from bacterial cell extracts, or through the use of recombinant
techniques by transformation, transfection, or infection of a host
cell with all or part of a fusion protein-encoding DNA fragment
such as a BA-05-encoding DNA fragment) with an antennapedia-derived
transport sequence in a suitable expression vehicle.
Example 20
Preparation of a Fusion Protein, BA-05
[0274] An example of a C3-like fusion protein is denoted
pGEX-4T/BA-05 (Seq ID NO: 4).
[0275] BA-05 is the name given herein to a protein made by ligating
a cDNA encoding C3 to a cDNA encoding a fusogenic 19-mer
peptide.
[0276] The method of example 19 can be used to prepare a fusion
protein, BA-05, which contains the following amino acid
sequence:
[0277] pGEX-4TBA-05 Protein Coding Sequence (SEQ ID NO:4)
TABLE-US-00014 Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr
Ser Ile Asn 1 5 10 15 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe
Thr Asn Ile Asp Gln 20 25 30 Ala Lys Ala Trp Gly Asn Ala Gln Tyr
Lys Lys Tyr Gly Leu Ser Lys 35 40 45 Ser Glu Lys Glu Ala Ile Val
Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55 60 Asn Gly Lys Leu Arg
Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser 65 70 75 80 Asn Leu Ile
Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95 Lys
Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105
110 Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile
115 120 125 Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn
Lys Asp 130 135 140 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met
Asn Val Ser Gln 145 150 155 160 Phe Ala Gly Arg Pro Ile Ile Thr Gln
Phe Lys Val Ala Lys Gly Ser 165 170 175 Lys Ala Gly Tyr Ile Asp Pro
Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185 190 Met Leu Leu Pro Arg
His Ser Thr Tyr His Ile Asp Asp Met Arg Leu 195 200 205 Ser Ser Asp
Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220 Ala
Ile Asn Pro Lys Glu Phe Val Met Glu Ser Arg Lys Arg Ala Arg 225 230
235 240 Gln Thr Tyr Thr Arg Tyr Gln Thr Leu Glu Leu Glu Lys Glu Phe
His 245 250 255 Phe Asn Arg Tyr Leu Thr Arg Arg Arg Arg Ile Glu Ile
Ala His Ala 260 265 270 Leu Cys Leu Thr Glu Arg Gln Ile Lys Ile Trp
Phe Gln Asn Arg Arg 275 280 285 Met Lys Trp Lys Lys Glu Asn 290
295
[0278] This C3-like fusion protein is prepared by the method
described to manipulate an antennapedia DNA into the pGEX4T/C3 DNA,
producing pGEX4T/BA-14. A clone with a frameshift mutation is
selected, and the protein is made and tested. When cultures test
positive despite the presence of a mutation, the plasmid DNA is
resequenced to confirm the mutation. The new clone is herein called
BA-05. To confirm the sequence of C3APLT, the coding sequence from
both strands are sequenced. The sequence for this clone is given in
Examples herein (nucleotide sequence of BA-05; SEQ ID NO:3, amino
acid sequence of BA-05; SEQ ID NO:4).
[0279] Another method useful to make BA-05 is to prepare
pGEX-4T/BA-14, then use the technique of site-directed mutagenesis
using two complementary oligonucleotide primers such as (SEQ ID
NO:58) 5' CCTAAAGAAT TCGTGATGAA TCCCGCAAAC GCGCA 3' and SEQ ID
NO:59 5' TGCGCGTTTG CGGGATTCAT CACGAATTCT TTAGG 3') containing a 1
basepair deletion in the pGEX4T-BA14 DNA. A QuikChange kit
(Statragene, LaJolla, Calif.) is used to incorporate the deletion
using extension of the primers in the presence of nucleotides. The
following cycle of temperatures is useful for preparation of BA-05:
1 cycle for 30 s at 95 C, then 18 cycles of 95 C for 30 s, 55 C for
1 min, and 68 C for 10.5 min, The DNA is then treated with the
restriction enzyme DpnI as described by the manufacturer. A portion
of the reaction is then transformed into E. coli DH5alpha or
XL1-Blue. Individual colonies of E. coli are isolated on agar
plates containing selective antibiotic, and grown in LB
medium+ampicillin. DNA is isolated using a MidiPrep Kit (Qiagen).
The DNA of 5 clones is sequenced and the sequence change is
confirmed. Protein is expressed from the DNA and purified as
described in Lehmann et al., 1999. The purified protein can be used
as a Rho antagonist in biological systems.
[0280] To prepare recombinant BA-05 (SEQ ID NO:3) the plasmids
containing the corresponding cDNA (pGEX-4T/BA-05) are transformed
into bacteria, strain XL-1 blue competent E. coli. The bacteria are
grown in L-broth (10 g/L Bacto-Tryptone, 5 g/L Yeast Extract, 10
g/L NaCl) with ampicillin at 50 ug/ml (BMC-Roche), in a shaking
incubator for 1 hr at 37.degree. C. and 300 rpm. Isopropyl
.beta.-D-thiogalactopyranoside (IPTG), (Gibco) is added to a final
concentration of 0.5 mM to induce the production of recombinant
protein and the culture is grown for a further 6 hours at
37.degree. C. and 250 rpm. Bacteria pellets ware obtained by
centrifugation in 250 ml centrifuge bottles at 7000 rpm for 6
minutes at 4.degree. C. Each pellet is re-suspended in 10 ml of
Buffer A (50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM MgCl..sub.2, 1 mM
DTT) plus 1 mM PMSF. All re-suspended pellets are pooled and
transferred to a 100 ml plastic beaker on ice. The remaining Buffer
A with PMSF is added to the pooled sample. The bacteria sample is
sonicated 6.times.20 seconds using a Branson Sonifier 450 probe
sonicator. Both the bacteria and probe are cooled on ice 1 minute
between sonications. The sonicate is centrifuged in a Sorvall SS-34
rotor at 16,000 rpm for 12 minutes at 4.degree. C. to clarify the
supernatant. The supernatant is transferred into fresh SS-34 tubes
and re-spun at 12,000 rpm for 12 minutes at 4.degree. C. Up to 20
ml of Glutathione-agarose beads (Sigma) are added to the cleared
lysate and placed on a rotating plate for 2 to 3 hours. The beads
are washed 4 times with buffer B, (Buffer A, NaCl is 150 mM, no
PSMF) then 2 times with Buffer C (Buffer B+2.5 mM CaCl.sub.2). The
final wash is poured out till the beads create a thick slurry. To
remove the glutathione S transferase sequence from the recombinant
protein, 20 U of Thrombin (Bovine, Plasminogen-free, Calbiochem) is
added, the beads are left on a rotator overnight at 4.degree. C.
After cleavage with thrombin the beads are loaded into an empty 20
ml column. Approximately 20 aliquots of 1 ml are collected by
elution with PBS. Samples of each aliquot of 0.5 ul are spotted on
nitrocellulose and stained with Amido Black to determine the
protein peak. Aliquots containing fusion proteins are pooled and
100 microliters of p-aminobenzamidine agarose beads (Sigma) are
added and left mixing for 45 minutes at 4.degree. C. This last step
removes the thrombin from the recombinant protein sample. The
recombinant protein is centrifuged to remove the beads and then
concentrated using a centriprep-10 concentrator (Amicon). The
concentrated recombinant protein is desalted with a PD-10 column
(Pharmacia, containing Sephadex G-25M) and ten 0.5 ml aliquots are
collected. A dot-blot is done on these samples to determine the
protein peak, and the appropriate aliquots pooled,
filter-sterilized, and stored at -80.degree. C. A protein assay (DC
assay, Biorad) is used to determine the concentration of
recombinant protein. Purity of the sample is determined by
SDS-PAGE, and bioactivity bioassay with NG-108 cells.
[0281] The products of this process can include fusion proteins
such as BA-14 as described in the general example, or new fusion
proteins produced by the cloning method that have properties such
as molecular weight and activity in Rho inactivation bioassays
different than the BA-14 molecule or control C3 protein, such as
BA-05. These new fusion proteins will contain the C3 sequence and
will be altered at the carboxyl terminus due to the method
employed.
Example 21
Preparation of Fusion Protein BA-07
[0282] The method of example 1 can be used to prepare a fusion
protein BA-07 which contains the following amino acid sequence:
TABLE-US-00015 Met Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser
Ile Asn Gln (SEQ ID NO: 57) 1 5 10 15 Lys Ala Tyr Ser Asn Thr Tyr
Gln Glu Phe Thr Asn Ile Asp Gln Ala 20 25 30 Lys Ala Trp Gly Asn
Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys Ser 35 40 45 Glu Lys Glu
Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile Asn 50 55 60 Gly
Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser Asn 65 70
75 80 Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met
Lys 85 90 95 Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro
Ala Tyr Leu 100 105 110 Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser
Asn Gly Thr Ile Asn 115 120 125 Lys Thr Ala Phe Glu Lys Ala Lys Ala
Lys Phe Leu Asn Lys Asp Arg 130 135 140 Leu Glu Tyr Gly Tyr Ile Ser
Thr Ser Leu Met Asn Val Ser Gln Phe 145 150 155 160 Ala Gly Arg Pro
Ile Ile Thr Lys Phe Lys Val Ala Lys Gly Ser Lys 165 170 175 Ala Gly
Tyr Ile Asp Pro Ile Ser Ala Phe Ala Gly Gln Leu Glu Met 180 185 190
Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu Ser 195
200 205 Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr
Ala 210 215 220 Ile Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala
Gln Gly Arg 225 230 235 240 His Thr Pro Gly Thr Arg Leu 245
[0283] Two PCR primers are designed to transfer one series of
recombinant constructs (BA-05) into the pET-9a vector (Novagen,
Madison, Wis.) to create BA-07 protein when expressed in an
appropriate expression system: Upper primer: 5'
GGATCTGGTTCCGCGTCATATGTCTAGAGTCGACCTG 3' (Seq ID NO: 38) Lower
primer: 5' CGCGGATCCATTAGTTCTCCTTCTTCCACTTC 3' (SEQ ID NO: 39). A
BamHI site at the 5' end of Seq ID NO: 39 is ggatccatta; the TGA is
replaced by TAAT (atta, in SEQ ID NO: 39).
[0284] A program useful to amplify the product using Pfu polymerase
comprises: 95.degree. C. 5' 1 cycle, then 94.degree. C.
2'.fwdarw.56.degree. C. 2'.fwdarw.70.degree. C. 2' 10 cycles, then
94.degree. C. 2'.fwdarw.70.degree. C. 3' 30 cycles and hold at
4.degree. C. A QIAEXII kit (Qiagen) can be used to purify an
agarose gel slice containing a desired DNA band. The insert and
vector are digested with BamHI and NdeI following the instructions
of the manufacturer (New England BioLabs, Beverly, Mass.), purified
using agarose gel electrophoresis and a QIAEXII kit (Qiagen), and
incubated together overnight with T4 DNA ligase following the
manufacturer's directions.
[0285] E. coli (DH5alpha, or preferably, XL 1-Blue) is transformed
with the ligation mixture. The clones can be checked by small-scale
induction and SDS-PAGE and can be assured by immunoblotting of the
crude lysates with anti-C3 antibody. Plasmid DNA is purified, and
can be assessed for purity. DNA sequencing can be performed (e.g.,
by LiCor technology in which the entire strand is sequenced for the
full length of the clone).
[0286] A first construct is prepared in this fashion (pET3a-BA-07,
SEQ ID NO:7) and acceptably matches the theoretical DNA sequence of
construct pGEX/BA-05 with a slight change in the 5' terminus due to
the cloning strategy.
[0287] A second construct, pET9a-BA-07, can be prepared by
subcloning the insert from pET3a-BA-07 into the pET9a vector by
cleaving the pET3a construct with BamHI and NdeI (New England
BioLabs, Beverly, Mass.) according to the manufacturers
instructions. pET9a plasmid DNA can be cleaved with the same
enzymes. The insert DNA and the vector DNA can be purified by
agarose gel electrophoresis. The insert can be ligated into the new
vector using T4 DNA ligase (New England BioLabs, Beverly, Mass.).
The ligated DNA can be transformed into DH5alpha cells and DNA can
be prepared using QIAGEN mini and maxi kits. Clones can be
characterized by restriction digestion and DNA sequencing of the
insert in both directions (e.g., BioS&T, Lachine, Quebec). The
construct DNA can be transformed into BL21 (DE3) cells,
BL21(DE3)/pLysS cells (Novagen, Madison, Wis.) or another suitable
expression system.
Example 22
General Method for Tritiated Thymidine Uptake as Measure of Cell
Proliferation
.sup.3H-Thymidine Incorporation Assays
[0288] Cell lines are tested for mycoplasma and found to be
negative prior to the initiation of the studies. Cell lines are
obtained from American Type Culture Collection (ATCC) (Rockville,
Md.). The line HEC-1B is cultured in Eagles Minimal Essential
Medium (E-MEM) supplemented with 10% fetal bovine serum (FBS) and
1% HEPES. The line Caco-2 is cultured in E-MEM supplemented with
20% FBS, 1% HEPES, 1 mM sodium pyruvate and 0.1 mM of non-essential
amino acid. The line SK-MEL-1 is cultured in Mc Coy's minimal
medium supplemented with 10% FBS and 1% HEPES. Volumes of 100 .mu.l
of each 2.times. working solution of C3-07, positive and vehicle
controls are plated in triplicate in 96-well microtiter plates
containing cells (4.times.10.sup.3/100 .mu.l), yielding a final
volume of 200 .mu.l. The plates were placed at 37.degree. C.
incubator with 100% humidity and 5% CO.sub.2. After 54 hours of
incubation, a volume of 20 .mu.l of tritiated thymidine
(3H-thymidine) (ICN, Montreal, Canada), containing 1.0 .mu.Ci, is
added to each well. The .sup.3H-thymidine is prepared in RPMI-1640
medium supplemented with 10% FBS. The cultures are incubated in the
same conditions as stated above, for a further 18 hours.
[0289] At the end of the incubation, the cells are harvested with
an automated cell harvester (Tomtec), and the incorporated counts
per minute (cpm) of .sup.3H-thymidine is measured with a microplate
scintillation counter (TopCount NXT, Packard). Values from the
wells treated with the BA-07 fusion protein are compared to values
of the vehicle control. Data is graphed with counts per minute
(cpm) on the y axis and the dose of fusion protein on the X axis.
Sequence CWU 1
1
59127DNAArtificial SequenceOligonucleotide used to remove the stop
codon from ADP-ribosyl transferase C3 (Clostridium botulinum) cDNA.
1gaattcttta ggattgatag ctgtgcc 27221DNAArtificial
SequenceOligonucleotide used to remove the stop codon from
ADP-ribosyl transferase C3 (Clostridium botulinum) cDNA.
2ggtggcgacc atcctccaaa a 213888DNAArtificial SequenceSequence of
C3APL includes ADP-ribosyl transferase C3 (Clostridium botulinum)
and Antennapedia sequence. 3gga tcc tct aga gtc gac ctg cag gca tgc
aat gct tat tcc att aat 48Gly Ser Ser Arg Val Asp Leu Gln Ala Cys
Asn Ala Tyr Ser Ile Asn1 5 10 15caa aag gct tat tca aat act tac cag
gag ttt act aat att gat caa 96Gln Lys Ala Tyr Ser Asn Thr Tyr Gln
Glu Phe Thr Asn Ile Asp Gln 20 25 30gca aaa gct tgg ggt aat gct cag
tat aaa aag tat gga cta agc aaa 144Ala Lys Ala Trp Gly Asn Ala Gln
Tyr Lys Lys Tyr Gly Leu Ser Lys 35 40 45tca gaa aaa gaa gct ata gta
tca tat act aaa agc gct agt gaa ata 192Ser Glu Lys Glu Ala Ile Val
Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55 60aat gga aag cta aga caa
aat aag gga gtt atc aat gga ttt cct tca 240Asn Gly Lys Leu Arg Gln
Asn Lys Gly Val Ile Asn Gly Phe Pro Ser65 70 75 80aat tta ata aaa
caa gtt gaa ctt tta gat aaa tct ttt aat aaa atg 288Asn Leu Ile Lys
Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95aag acc cct
gaa aat att atg tta ttt aga ggc gac gac cct gct tat 336Lys Thr Pro
Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110tta
gga aca gaa ttt caa aac act ctt ctt aat tca aat ggt aca att 384Leu
Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120
125aat aaa acg gct ttt gaa aag gct aaa gct aag ttt tta aat aaa gat
432Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp
130 135 140aga ctt gaa tat gga tat att agt act tca tta atg aat gtc
tct caa 480Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val
Ser Gln145 150 155 160ttt gca gga aga cca att att aca caa ttt aaa
gta gca aaa ggc tca 528Phe Ala Gly Arg Pro Ile Ile Thr Gln Phe Lys
Val Ala Lys Gly Ser 165 170 175aag gca gga tat att gac cct att agt
gct ttt cag gga caa ctt gaa 576Lys Ala Gly Tyr Ile Asp Pro Ile Ser
Ala Phe Gln Gly Gln Leu Glu 180 185 190atg ttg ctt cct aga cat agt
act tat cat ata gac gat atg aga ttg 624Met Leu Leu Pro Arg His Ser
Thr Tyr His Ile Asp Asp Met Arg Leu 195 200 205tct tct gat ggt aaa
caa ata ata att aca gca aca atg atg ggc aca 672Ser Ser Asp Gly Lys
Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220gct atc aat
cct aaa gaa ttc gtg atg gaa tcc cgc aaa cgc gca agg 720Ala Ile Asn
Pro Lys Glu Phe Val Met Glu Ser Arg Lys Arg Ala Arg225 230 235
240cag aca tac acc cgg tac cag act cta gag cta gag aag gag ttt cac
768Gln Thr Tyr Thr Arg Tyr Gln Thr Leu Glu Leu Glu Lys Glu Phe His
245 250 255ttc aat cgc tac ttg acc cgt cgg cga agg atc gag atc gcc
cac gcc 816Phe Asn Arg Tyr Leu Thr Arg Arg Arg Arg Ile Glu Ile Ala
His Ala 260 265 270ctg tgc ctc acg gag cgc cag ata aag att tgg ttc
cag aat cgg cgc 864Leu Cys Leu Thr Glu Arg Gln Ile Lys Ile Trp Phe
Gln Asn Arg Arg 275 280 285atg aag tgg aag aag gag aac tga 888Met
Lys Trp Lys Lys Glu Asn 290 2954295PRTArtificial SequenceSequence
of C3APLT includes ADP-ribosyl transferase C3 (Clostridium
botulinum) and Antennapedia sequence. 4Gly Ser Ser Arg Val Asp Leu
Gln Ala Cys Asn Ala Tyr Ser Ile Asn1 5 10 15Gln Lys Ala Tyr Ser Asn
Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln 20 25 30Ala Lys Ala Trp Gly
Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 35 40 45Ser Glu Lys Glu
Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55 60Asn Gly Lys
Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser65 70 75 80Asn
Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90
95Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr
100 105 110Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly
Thr Ile 115 120 125Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe
Leu Asn Lys Asp 130 135 140Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser
Leu Met Asn Val Ser Gln145 150 155 160Phe Ala Gly Arg Pro Ile Ile
Thr Gln Phe Lys Val Ala Lys Gly Ser 165 170 175Lys Ala Gly Tyr Ile
Asp Pro Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185 190Met Leu Leu
Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu 195 200 205Ser
Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 210 215
220Ala Ile Asn Pro Lys Glu Phe Val Met Glu Ser Arg Lys Arg Ala
Arg225 230 235 240Gln Thr Tyr Thr Arg Tyr Gln Thr Leu Glu Leu Glu
Lys Glu Phe His 245 250 255Phe Asn Arg Tyr Leu Thr Arg Arg Arg Arg
Ile Glu Ile Ala His Ala 260 265 270Leu Cys Leu Thr Glu Arg Gln Ile
Lys Ile Trp Phe Gln Asn Arg Arg 275 280 285Met Lys Trp Lys Lys Glu
Asn 290 2955774DNAArtificial SequenceSequence of C3APS Includes
ADP-ribosyl transferase C3 (Clostridium botulinum) and Antennapedia
sequence. 5gga tcc tct aga gtc gac ctg cag gca tgc aat gct tat tcc
att aat 48Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser
Ile Asn1 5 10 15caa aag gct tat tca aat act tac cag gag ttt act aat
att gat caa 96Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn
Ile Asp Gln 20 25 30gca aaa gct tgg ggt aat gct cag tat aaa aag tat
gga cta agc aaa 144Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr
Gly Leu Ser Lys 35 40 45tca gaa aaa gaa gct ata gta tca tat act aaa
agc gct agt gaa ata 192Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 50 55 60aat gga aag cta aga caa aat aag gga gtt
atc aat gga ttt cct tca 240Asn Gly Lys Leu Arg Gln Asn Lys Gly Val
Ile Asn Gly Phe Pro Ser65 70 75 80aat tta ata aaa caa gtt gaa ctt
tta gat aaa tct ttt aat aaa atg 288Asn Leu Ile Lys Gln Val Glu Leu
Leu Asp Lys Ser Phe Asn Lys Met 85 90 95aag acc cct gaa aat att atg
tta ttt aga ggc gac gac cct gct tat 336Lys Thr Pro Glu Asn Ile Met
Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110tta gga aca gaa ttt
caa aac act ctt ctt aat tca aat ggt aca att 384Leu Gly Thr Glu Phe
Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125aat aaa acg
gct ttt gaa aag gct aaa gct aag ttt tta aat aaa gat 432Asn Lys Thr
Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140aga
ctt gaa tat gga tat att agt act tca tta atg aat gtc tct caa 480Arg
Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln145 150
155 160ttt gca gga aga cca att att aca caa ttt aaa gta gca aaa ggc
tca 528Phe Ala Gly Arg Pro Ile Ile Thr Gln Phe Lys Val Ala Lys Gly
Ser 165 170 175aag gca gga tat att gac cct att agt gct ttt cag gga
caa ctt gaa 576Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Gln Gly
Gln Leu Glu 180 185 190atg ttg ctt cct aga cat agt act tat cat ata
gac gat atg aga ttg 624Met Leu Leu Pro Arg His Ser Thr Tyr His Ile
Asp Asp Met Arg Leu 195 200 205tct tct gat ggt aaa caa ata ata att
aca gca aca atg atg ggc aca 672Ser Ser Asp Gly Lys Gln Ile Ile Ile
Thr Ala Thr Met Met Gly Thr 210 215 220gct atc aat cct aaa gaa ttc
cgc cag atc aag att tgg ttc cag aat 720Ala Ile Asn Pro Lys Glu Phe
Arg Gln Ile Lys Ile Trp Phe Gln Asn225 230 235 240cgt cgc atg aag
tgg aag aag gtc gac tcg agc ggc cgc atc gtg act 768Arg Arg Met Lys
Trp Lys Lys Val Asp Ser Ser Gly Arg Ile Val Thr 245 250 255gac tga
774Asp6257PRTArtificial SequenceSequence of C3APS Includes
ADP-ribosyl transferase C3 (Clostridium botulinum) and Antennapedia
sequence. 6Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser
Ile Asn1 5 10 15Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn
Ile Asp Gln 20 25 30Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr
Gly Leu Ser Lys 35 40 45Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 50 55 60Asn Gly Lys Leu Arg Gln Asn Lys Gly Val
Ile Asn Gly Phe Pro Ser65 70 75 80Asn Leu Ile Lys Gln Val Glu Leu
Leu Asp Lys Ser Phe Asn Lys Met 85 90 95Lys Thr Pro Glu Asn Ile Met
Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110Leu Gly Thr Glu Phe
Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125Asn Lys Thr
Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140Arg
Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln145 150
155 160Phe Ala Gly Arg Pro Ile Ile Thr Gln Phe Lys Val Ala Lys Gly
Ser 165 170 175Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Gln Gly
Gln Leu Glu 180 185 190Met Leu Leu Pro Arg His Ser Thr Tyr His Ile
Asp Asp Met Arg Leu 195 200 205Ser Ser Asp Gly Lys Gln Ile Ile Ile
Thr Ala Thr Met Met Gly Thr 210 215 220Ala Ile Asn Pro Lys Glu Phe
Arg Gln Ile Lys Ile Trp Phe Gln Asn225 230 235 240Arg Arg Met Lys
Trp Lys Lys Val Asp Ser Ser Gly Arg Ile Val Thr 245 250
255Asp724DNAArtificial SequenceOligonucleotide used in the
amplification of Antennapedia sequence 7gaatcccgca aacgcgcaag gcag
24827DNAArtificial SequenceOligonucleotide used in the
amplification of Antennapedia sequence 8tcagttctcc ttcttccact
tcatgcg 27954DNAArtificial SequenceOligonucleotide used in the
cloning of sequences from Antennapedia 9aattccgcca gatcaagatt
tggttccaga atcgtcgcat gaagtggaag aagg 541054DNAArtificial
SequenceOligonucleotide used in the cloning of sequences from
Antennapedia 10ggcggtctag ttctaaacca agctcttagc agcgtagttc
accttcttcc agct 541126DNAArtificial SequenceOligonucleotide used
inthe amplification of a sequence corresponding to amino acid 27-72
of HIV-1 Tat 11gaatccaagc atccaggaag tcagcc 261221DNAArtificial
SequenceOligonucleotide used inthe amplification of a sequence
corresponding to amino acid 27-72 of HIV-1 Tat 12accagccacc
accttctgat a 2113876DNAArtificial SequenceSequence of C3-TL
Includes ADP-ribosyl transferase C3 (Clostridium botulinum) and
HIV-1 Tat sequence. 13gga tcc tct aga gtc gac ctg cag gca tgc aat
gct tat tcc att aat 48Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn
Ala Tyr Ser Ile Asn1 5 10 15caa aag gct tat tca aat act tac cag gag
ttt act aat att gat caa 96Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu
Phe Thr Asn Ile Asp Gln 20 25 30gca aaa gct tgg ggt aat gct cag tat
aaa aag tat gga cta agc aaa 144Ala Lys Ala Trp Gly Asn Ala Gln Tyr
Lys Lys Tyr Gly Leu Ser Lys 35 40 45tca gaa aaa gaa gct ata gta tca
tat act aaa agc gct agt gaa ata 192Ser Glu Lys Glu Ala Ile Val Ser
Tyr Thr Lys Ser Ala Ser Glu Ile 50 55 60aat gga aag cta aga caa aat
aag gga gtt atc aat gga ttt cct tca 240Asn Gly Lys Leu Arg Gln Asn
Lys Gly Val Ile Asn Gly Phe Pro Ser65 70 75 80aat tta ata aaa caa
gtt gaa ctt tta gat aaa tct ttt aat aaa atg 288Asn Leu Ile Lys Gln
Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95aag acc cct gaa
aat att atg tta ttt aga ggc gac gac cct gct tat 336Lys Thr Pro Glu
Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110tta gga
aca gaa ttt caa aac act ctt ctt aat tca aat ggt aca att 384Leu Gly
Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120
125aat aaa acg gct ttt gaa aag gct aaa gct aag ttt tta aat aaa gat
432Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp
130 135 140aga ctt gaa tat gga tat att agt act tca tta atg aat gtc
tct caa 480Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val
Ser Gln145 150 155 160ttt gca gga aga cca att att aca caa ttt aaa
gta gca aaa ggc tca 528Phe Ala Gly Arg Pro Ile Ile Thr Gln Phe Lys
Val Ala Lys Gly Ser 165 170 175aag gca gga tat att gac cct att agt
gct ttt cag gga caa ctt gaa 576Lys Ala Gly Tyr Ile Asp Pro Ile Ser
Ala Phe Gln Gly Gln Leu Glu 180 185 190atg ttg ctt cct aga cat agt
act tat cat ata gac gat atg aga ttg 624Met Leu Leu Pro Arg His Ser
Thr Tyr His Ile Asp Asp Met Arg Leu 195 200 205tct tct gat ggt aaa
caa ata ata att aca gca aca atg atg ggc aca 672Ser Ser Asp Gly Lys
Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220gct atc aat
cct aaa gaa ttc aag cat cca gga agt cag cct aaa act 720Ala Ile Asn
Pro Lys Glu Phe Lys His Pro Gly Ser Gln Pro Lys Thr225 230 235
240gct tgt acc aat tgc tat tgt aaa aag tgt tgc ttt cat tgc caa gtt
768Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln Val
245 250 255tgt ttc ata aca aaa gcc tta ggc atc tcc tat ggc agg aag
cgg aga 816Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg Lys
Arg Arg 260 265 270cag cga cga aga gct cat cag aac agt cag act cat
caa gct tct cta 864Gln Arg Arg Arg Ala His Gln Asn Ser Gln Thr His
Gln Ala Ser Leu 275 280 285tca aag cag taa 876Ser Lys Gln
29014291PRTArtificial SequenceSequence of C3-TL Includes
ADP-ribosyl transferase C3 (Clostridium botulinum) and HIV-1 Tat
sequence. 14Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser
Ile Asn1 5 10 15Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn
Ile Asp Gln 20 25 30Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr
Gly Leu Ser Lys 35 40 45Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 50 55 60Asn Gly Lys Leu Arg Gln Asn Lys Gly Val
Ile Asn Gly Phe Pro Ser65 70 75 80Asn Leu Ile Lys Gln Val Glu Leu
Leu Asp Lys Ser Phe Asn Lys Met 85 90 95Lys Thr Pro Glu Asn Ile Met
Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110Leu Gly Thr Glu Phe
Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125Asn Lys Thr
Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140Arg
Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln145 150
155 160Phe Ala Gly Arg Pro Ile Ile Thr Gln Phe Lys Val Ala Lys Gly
Ser 165 170 175Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Gln Gly
Gln Leu Glu 180 185 190Met Leu Leu Pro Arg His Ser Thr Tyr His Ile
Asp Asp Met Arg Leu 195 200 205Ser Ser Asp Gly Lys Gln Ile Ile Ile
Thr Ala Thr
Met Met Gly Thr 210 215 220Ala Ile Asn Pro Lys Glu Phe Lys His Pro
Gly Ser Gln Pro Lys Thr225 230 235 240Ala Cys Thr Asn Cys Tyr Cys
Lys Lys Cys Cys Phe His Cys Gln Val 245 250 255Cys Phe Ile Thr Lys
Ala Leu Gly Ile Ser Tyr Gly Arg Lys Arg Arg 260 265 270Gln Arg Arg
Arg Ala His Gln Asn Ser Gln Thr His Gln Ala Ser Leu 275 280 285Ser
Lys Gln 2901539DNAArtificial SequenceOligonucleotide used in the
cloning of sequences from HIV-1 Tat 15aattctatgg tcgtaaaaaa
cgtcgtcaac gtcgtcgtg 391639DNAArtificial SequenceOligonucleotide
used in the cloning of sequences from HIV-1 Tat 16gataccagca
ttttttgcag cagttgcagc agcacagct 3917756DNAArtificial
SequenceSequence of C3-TS Includes ADP-ribosyl transferase C3
(Clostridium botulinum) and HIV-1 Tat sequence. 17gga tcc tct aga
gtc gac ctg cag gca tgc aat gct tat tcc att aat 48Gly Ser Ser Arg
Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn1 5 10 15caa aag gct
tat tca aat act tac cag gag ttt act aat att gat caa 96Gln Lys Ala
Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln 20 25 30gca aaa
gct tgg ggt aat gct cag tat aaa aag tat gga cta agc aaa 144Ala Lys
Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 35 40 45tca
gaa aaa gaa gct ata gta tca tat act aaa agc gct agt gaa ata 192Ser
Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55
60aat gga aag cta aga caa aat aag gga gtt atc aat gga ttt cct tca
240Asn Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro
Ser65 70 75 80aat tta ata aaa caa gtt gaa ctt tta gat aaa tct ttt
aat aaa atg 288Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe
Asn Lys Met 85 90 95aag acc cct gaa aat att atg tta ttt aga ggc gac
gac cct gct tat 336Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp
Asp Pro Ala Tyr 100 105 110tta gga aca gaa ttt caa aac act ctt ctt
aat tca aat ggt aca att 384Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu
Asn Ser Asn Gly Thr Ile 115 120 125aat aaa acg gct ttt gaa aag gct
aaa gct aag ttt tta aat aaa gat 432Asn Lys Thr Ala Phe Glu Lys Ala
Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140aga ctt gaa tat gga tat
att agt act tca tta atg aat gtc tct caa 480Arg Leu Glu Tyr Gly Tyr
Ile Ser Thr Ser Leu Met Asn Val Ser Gln145 150 155 160ttt gca gga
aga cca att att aca caa ttt aaa gta gca aaa ggc tca 528Phe Ala Gly
Arg Pro Ile Ile Thr Gln Phe Lys Val Ala Lys Gly Ser 165 170 175aag
gca gga tat att gac cct att agt gct ttt cag gga caa ctt gaa 576Lys
Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185
190atg ttg ctt cct aga cat agt act tat cat ata gac gat atg aga ttg
624Met Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu
195 200 205tct tct gat ggt aaa caa ata ata att aca gca aca atg atg
ggc aca 672Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met
Gly Thr 210 215 220gct atc aat cct aaa gaa ttc tat ggt gct aaa aaa
cgt cgt caa cgt 720Ala Ile Asn Pro Lys Glu Phe Tyr Gly Ala Lys Lys
Arg Arg Gln Arg225 230 235 240cgt cgt gtc gac tcg agc ggc ccg cat
cgt gac tga 756Arg Arg Val Asp Ser Ser Gly Pro His Arg Asp 245
25018251PRTArtificial SequenceSequence of C3-TS Includes
ADP-ribosyl transferase C3 (Clostridium botulinum) and HIV-1 Tat
sequence. 18Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser
Ile Asn1 5 10 15Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn
Ile Asp Gln 20 25 30Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr
Gly Leu Ser Lys 35 40 45Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 50 55 60Asn Gly Lys Leu Arg Gln Asn Lys Gly Val
Ile Asn Gly Phe Pro Ser65 70 75 80Asn Leu Ile Lys Gln Val Glu Leu
Leu Asp Lys Ser Phe Asn Lys Met 85 90 95Lys Thr Pro Glu Asn Ile Met
Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110Leu Gly Thr Glu Phe
Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125Asn Lys Thr
Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140Arg
Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln145 150
155 160Phe Ala Gly Arg Pro Ile Ile Thr Gln Phe Lys Val Ala Lys Gly
Ser 165 170 175Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Gln Gly
Gln Leu Glu 180 185 190Met Leu Leu Pro Arg His Ser Thr Tyr His Ile
Asp Asp Met Arg Leu 195 200 205Ser Ser Asp Gly Lys Gln Ile Ile Ile
Thr Ala Thr Met Met Gly Thr 210 215 220Ala Ile Asn Pro Lys Glu Phe
Tyr Gly Ala Lys Lys Arg Arg Gln Arg225 230 235 240Arg Arg Val Asp
Ser Ser Gly Pro His Arg Asp 245 250191413DNAArtificial
SequenceIncludes GST sequences, ADP-ribosyl transferase C3 (C.
botulinum) sequence and a random basic amino acid sequence. 19atg
tcc cct ata cta ggt tat tgg aaa att aag ggc ctt gtg caa ccc 48Met
Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro1 5 10
15act cga ctt ctt ttg gaa tat ctt gaa gaa aaa tat gaa gag cat ttg
96Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30tat gag cgc gat gaa ggt gat aaa tgg cga aac aaa aag ttt gaa
ttg 144Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu
Leu 35 40 45ggt ttg gag ttt ccc aat ctt cct tat tat att gat ggt gat
gtt aaa 192Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp
Val Lys 50 55 60tta aca cag tct atg gcc atc ata cgt tat ata gct gac
aag cac aac 240Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp
Lys His Asn65 70 75 80atg ttg ggt ggt tgt cca aaa gag cgt gca gag
att tca atg ctt gaa 288Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu
Ile Ser Met Leu Glu 85 90 95gga gcg gtt ttg gat att aga tac ggt gtt
tcg aga att gca tat agt 336Gly Ala Val Leu Asp Ile Arg Tyr Gly Val
Ser Arg Ile Ala Tyr Ser 100 105 110aaa gac ttt gaa act ctc aaa gtt
gat ttt ctt agc aag cta cct gaa 384Lys Asp Phe Glu Thr Leu Lys Val
Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125atg ctg aaa atg ttc gaa
gat cgt tta tgt cat aaa aca tat tta aat 432Met Leu Lys Met Phe Glu
Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140ggt gat cat gta
acc cat cct gac ttc atg ttg tat gac gct ctt gat 480Gly Asp His Val
Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150 155 160gtt
gtt tta tac atg gac cca atg tgc ctg gat gcg ttc cca aaa tta 528Val
Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170
175gtt tgt ttt aaa aaa cgt att gaa gct atc cca caa att gat aag tac
576Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190ttg aaa tcc agc aag tat ata gca tgg cct ttg cag ggc tgg
caa gcc 624Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp
Gln Ala 195 200 205acg ttt ggt ggt ggc gac cat cct cca aaa tcg gat
ctg gtt ccg cgt 672Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp
Leu Val Pro Arg 210 215 220gga tcc tct aga gtc gac ctg cag gca tgc
aat gct tat tcc att aat 720Gly Ser Ser Arg Val Asp Leu Gln Ala Cys
Asn Ala Tyr Ser Ile Asn225 230 235 240caa aag gct tat tca aat act
tac cag gag ttt act aat att gat caa 768Gln Lys Ala Tyr Ser Asn Thr
Tyr Gln Glu Phe Thr Asn Ile Asp Gln 245 250 255gca aaa gct tgg ggt
aat gct cag tat aaa aag tat gga cta agc aaa 816Ala Lys Ala Trp Gly
Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 260 265 270tca gaa aaa
gaa gct ata gta tca tat act aaa agc gct agt gaa ata 864Ser Glu Lys
Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 275 280 285aat
gga aag cta aga caa aat aag gga gtt atc aat gga ttt cct tca 912Asn
Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser 290 295
300aat tta ata aaa caa gtt gaa ctt tta gat aaa tct ttt aat aaa atg
960Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys
Met305 310 315 320aag acc cct gaa aat att atg tta ttt aga ggc gac
gac cct gct tat 1008Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp
Asp Pro Ala Tyr 325 330 335tta gga aca gaa ttt caa aac act ctt ctt
aat tca aat ggt aca att 1056Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu
Asn Ser Asn Gly Thr Ile 340 345 350aat aaa acg gct ttt gaa aag gct
aaa gct aag ttt tta aat aaa gat 1104Asn Lys Thr Ala Phe Glu Lys Ala
Lys Ala Lys Phe Leu Asn Lys Asp 355 360 365aga ctt gaa tat gga tat
att agt act tca tta atg aat gtt tct caa 1152Arg Leu Glu Tyr Gly Tyr
Ile Ser Thr Ser Leu Met Asn Val Ser Gln 370 375 380ttt gca gga aga
cca att att aca aaa ttt aaa gta gca aaa ggc tca 1200Phe Ala Gly Arg
Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly Ser385 390 395 400aag
gca gga tat att gac cct att agt gct ttt cag gga caa ctt gaa 1248Lys
Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Gln Gly Gln Leu Glu 405 410
415atg ttg ctt cct aga cat agt act tat cat ata gac gat atg aga ttg
1296Met Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu
420 425 430tct tct gat ggt aaa caa ata ata att aca gca aca atg atg
ggc aca 1344Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met
Gly Thr 435 440 445gct atc aat cct aaa gaa ttc aga agg aaa caa aga
aga aaa aga aga 1392Ala Ile Asn Pro Lys Glu Phe Arg Arg Lys Gln Arg
Arg Lys Arg Arg 450 455 460ctg cag gcg gcc gca tcg tga 1413Leu Gln
Ala Ala Ala Ser465 47020470PRTArtificial SequenceIncludes GST
sequences, ADP-ribosyl transferase C3 (C. botulinum) sequence and a
random basic amino acid sequence 20Met Ser Pro Ile Leu Gly Tyr Trp
Lys Ile Lys Gly Leu Val Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr
Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly
Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro
Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser
Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn65 70 75 80Met Leu
Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly
Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105
110Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr
Leu Asn 130 135 140Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr
Asp Ala Leu Asp145 150 155 160Val Val Leu Tyr Met Asp Pro Met Cys
Leu Asp Ala Phe Pro Lys Leu 165 170 175Val Cys Phe Lys Lys Arg Ile
Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys
Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly
Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg 210 215 220Gly
Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn225 230
235 240Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp
Gln 245 250 255Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly
Leu Ser Lys 260 265 270Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 275 280 285Asn Gly Lys Leu Arg Gln Asn Lys Gly
Val Ile Asn Gly Phe Pro Ser 290 295 300Asn Leu Ile Lys Gln Val Glu
Leu Leu Asp Lys Ser Phe Asn Lys Met305 310 315 320Lys Thr Pro Glu
Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 325 330 335Leu Gly
Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 340 345
350Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp
355 360 365Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val
Ser Gln 370 375 380Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val
Ala Lys Gly Ser385 390 395 400Lys Ala Gly Tyr Ile Asp Pro Ile Ser
Ala Phe Gln Gly Gln Leu Glu 405 410 415Met Leu Leu Pro Arg His Ser
Thr Tyr His Ile Asp Asp Met Arg Leu 420 425 430Ser Ser Asp Gly Lys
Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 435 440 445Ala Ile Asn
Pro Lys Glu Phe Arg Arg Lys Gln Arg Arg Lys Arg Arg 450 455 460Leu
Gln Ala Ala Ala Ser465 4702116PRTArtificial SequenceRandom basic
amino acid sequence of C3Basic1 21Lys Arg Arg Arg Arg Arg Pro Lys
Lys Arg Arg Arg Ala Lys Arg Arg1 5 10 152248DNAArtificial
SequenceOligonucleotide used in the cloning of a random basic amino
acid sequence in C3Basic1 22aagagaaggc gaagaagacc taagaagaga
cgaagggcga agaggaga 482348DNAArtificial SequenceOligonucleotide
used in the cloning of a random basic amino acid sequence in
C3Basic1 23ttctcttccg cttcttctgg attcttctct gcttcccgct tctcctct
4824792DNAArtificial SequenceSequence of C3Basic1 includes
ADP-ribosyl transferase C3 (Clostridium botulinum) sequence and a
sequence encoding a random basic amino acid sequence and a
Histidine tag. 24gga tcc tct aga gtc gac ctg cag gca tgc aat gct
tat tcc att aat 48Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala
Tyr Ser Ile Asn1 5 10 15caa aag gct tat tca aat act tac cag gag ttt
act aat att gat caa 96Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe
Thr Asn Ile Asp Gln 20 25 30gca aaa gct tgg ggt aat gct cag tat aaa
aag tat gga cta agc aaa 144Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys
Lys Tyr Gly Leu Ser Lys 35 40 45tca gaa aaa gaa gct ata gta tca tat
act aaa agc gct agt gaa ata 192Ser Glu Lys Glu Ala Ile Val Ser Tyr
Thr Lys Ser Ala Ser Glu Ile 50 55 60aat gga aag cta aga caa aat aag
gga gtt atc aat gga ttt cct tca 240Asn Gly Lys Leu Arg Gln Asn Lys
Gly Val Ile Asn Gly Phe Pro Ser65 70 75 80aat tta ata aaa caa gtt
gaa ctt tta gat aaa tct ttt aat aaa atg 288Asn Leu Ile Lys Gln Val
Glu Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95aag acc cct gaa aat
att atg tta ttt aga ggc gac gac cct gct tat 336Lys Thr Pro Glu Asn
Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110tta gga aca
gaa ttt caa aac act ctt ctt aat tca aat ggt aca att 384Leu Gly Thr
Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125aat
aaa acg gct ttt gaa aag gct aaa gct aag ttt tta aat aaa gat 432Asn
Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135
140aga ctt gaa tat gga tat att agt act tca tta atg aat gtt tct caa
480Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser
Gln145 150 155 160ttt gca gga aga cca att att aca aaa ttt aaa gta
gca aaa ggc tca 528Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val
Ala Lys Gly Ser
165 170 175aag gca gga tat att gac cct att agt gct ttt cag gga caa
ctt gaa 576Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Gln Gly Gln
Leu Glu 180 185 190atg ttg ctt cct aga cat agt act tat cat ata gac
gat atg aga ttg 624Met Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp
Asp Met Arg Leu 195 200 205tct tct gat ggt aaa caa ata ata att aca
gca aca atg atg ggc aca 672Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr
Ala Thr Met Met Gly Thr 210 215 220gct atc aat cct aaa gaa ttc aag
aga agg cga aga aga cct aag aag 720Ala Ile Asn Pro Lys Glu Phe Lys
Arg Arg Arg Arg Arg Pro Lys Lys225 230 235 240aga cga agg gcg aag
agg aga cac cac cac cac cac cac gtc gac tcg 768Arg Arg Arg Ala Lys
Arg Arg His His His His His His Val Asp Ser 245 250 255agc ggc cgc
atc gtg act gac tga 792Ser Gly Arg Ile Val Thr Asp
26025263PRTArtificial SequenceSequence of C3Basic1 includes
ADP-ribosyl transferase C3 (Clostridium botulinum) sequence and a
sequence encoding a random basic amino acid sequence and a
Histidine tag. 25Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala
Tyr Ser Ile Asn1 5 10 15Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe
Thr Asn Ile Asp Gln 20 25 30Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys
Lys Tyr Gly Leu Ser Lys 35 40 45Ser Glu Lys Glu Ala Ile Val Ser Tyr
Thr Lys Ser Ala Ser Glu Ile 50 55 60Asn Gly Lys Leu Arg Gln Asn Lys
Gly Val Ile Asn Gly Phe Pro Ser65 70 75 80Asn Leu Ile Lys Gln Val
Glu Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95Lys Thr Pro Glu Asn
Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110Leu Gly Thr
Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125Asn
Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135
140Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser
Gln145 150 155 160Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val
Ala Lys Gly Ser 165 170 175Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala
Phe Gln Gly Gln Leu Glu 180 185 190Met Leu Leu Pro Arg His Ser Thr
Tyr His Ile Asp Asp Met Arg Leu 195 200 205Ser Ser Asp Gly Lys Gln
Ile Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220Ala Ile Asn Pro
Lys Glu Phe Lys Arg Arg Arg Arg Arg Pro Lys Lys225 230 235 240Arg
Arg Arg Ala Lys Arg Arg His His His His His His Val Asp Ser 245 250
255Ser Gly Arg Ile Val Thr Asp 2602613PRTArtificial SequenceRandom
amino acid sequence of C3Basic2 26Lys Arg Arg Arg Arg Lys Lys Arg
Arg Gln Arg Arg Arg1 5 102739DNAArtificial SequenceOligonucleotide
used in the cloning of a random basic amino acid sequence in
C3Basic2 27aagcgtcgac gtagaaagaa acgtagacag cgtagacgt
392839DNAArtificial SequenceOligonucleotide used in the cloning of
a random basic amino acid sequence in C3Basic2 28ttcgcagctg
catctttctt tgcatctgtc gcatctgca 3929783DNAArtificial
SequenceSequence of C3Basic2 includes sequences from ADP-ribosyl-
transferase C3 (Clostridium botulinum) and a sequence encoding a
random basic amino acid sequence and a histidine tag. 29gga tcc tct
aga gtc gac ctg cag gca tgc aat gct tat tcc att aat 48Gly Ser Ser
Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn1 5 10 15caa aag
gct tat tca aat act tac cag gag ttt act aat att gat caa 96Gln Lys
Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln 20 25 30gca
aaa gct tgg ggt aat gct cag tat aaa aag tat gga cta agc aaa 144Ala
Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 35 40
45tca gaa aaa gaa gct ata gta tca tat act aaa agc gct agt gaa ata
192Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile
50 55 60aat gga aag cta aga caa aat aag gga gtt atc aat gga ttt cct
tca 240Asn Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro
Ser65 70 75 80aat tta ata aaa caa gtt gaa ctt tta gat aaa tct ttt
aat aaa atg 288Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe
Asn Lys Met 85 90 95aag acc cct gaa aat att atg tta ttt aga ggc gac
gac cct gct tat 336Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp
Asp Pro Ala Tyr 100 105 110tta gga aca gaa ttt caa aac act ctt ctt
aat tca aat ggt aca att 384Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu
Asn Ser Asn Gly Thr Ile 115 120 125aat aaa acg gct ttt gaa aag gct
aaa gct aag ttt tta aat aaa gat 432Asn Lys Thr Ala Phe Glu Lys Ala
Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140aga ctt gaa tat gga tat
att agt act tca tta atg aat gtt tct caa 480Arg Leu Glu Tyr Gly Tyr
Ile Ser Thr Ser Leu Met Asn Val Ser Gln145 150 155 160ttt gca gga
aga cca att att aca aaa ttt aaa gta gca aaa ggc tca 528Phe Ala Gly
Arg Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly Ser 165 170 175aag
gca gga tat att gac cct att agt gct ttt cag gga caa ctt gaa 576Lys
Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185
190atg ttg ctt cct aga cat agt act tat cat ata gac gat atg aga ttg
624Met Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu
195 200 205tct tct gat ggt aaa caa ata ata att aca gca aca atg atg
ggc aca 672Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met
Gly Thr 210 215 220gct atc aat cct aaa gaa ttc aag cgt cga cgt aga
aag aaa cgt aga 720Ala Ile Asn Pro Lys Glu Phe Lys Arg Arg Arg Arg
Lys Lys Arg Arg225 230 235 240cag cgt aga cgt cac cac cac cac cac
cac gtc gac tcg agc ggc cgc 768Gln Arg Arg Arg His His His His His
His Val Asp Ser Ser Gly Arg 245 250 255atc gtg act gac tga 783Ile
Val Thr Asp 26030260PRTArtificial SequenceSequence of C3Basic2
includes sequences from ADP-ribosyl- transferase C3 (Clostridium
botulinum) and a sequence encoding a random basic amino acid
sequence and a histidine tag. 30Gly Ser Ser Arg Val Asp Leu Gln Ala
Cys Asn Ala Tyr Ser Ile Asn1 5 10 15Gln Lys Ala Tyr Ser Asn Thr Tyr
Gln Glu Phe Thr Asn Ile Asp Gln 20 25 30Ala Lys Ala Trp Gly Asn Ala
Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 35 40 45Ser Glu Lys Glu Ala Ile
Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55 60Asn Gly Lys Leu Arg
Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser65 70 75 80Asn Leu Ile
Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95Lys Thr
Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105
110Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile
115 120 125Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn
Lys Asp 130 135 140Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met
Asn Val Ser Gln145 150 155 160Phe Ala Gly Arg Pro Ile Ile Thr Lys
Phe Lys Val Ala Lys Gly Ser 165 170 175Lys Ala Gly Tyr Ile Asp Pro
Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185 190Met Leu Leu Pro Arg
His Ser Thr Tyr His Ile Asp Asp Met Arg Leu 195 200 205Ser Ser Asp
Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220Ala
Ile Asn Pro Lys Glu Phe Lys Arg Arg Arg Arg Lys Lys Arg Arg225 230
235 240Gln Arg Arg Arg His His His His His His Val Asp Ser Ser Gly
Arg 245 250 255Ile Val Thr Asp 260319PRTArtificial SequenceReverse
HIV-1 Tat amino acid sequence of C3Basic3 31Arg Arg Lys Gln Arg Arg
Lys Arg Arg1 53227DNAArtificial SequenceOligonucleotide used in the
cloning of a reverse HIV Tat sequence in C3Basic3 32agaaggaaac
aaagaagaaa aagaaga 273327DNAArtificial SequenceOligonucleotide used
in the cloning of a reverse HIV Tat sequence in C3Basic3
33tcttcctttg tttcttcttt ttcttct 2734771DNAArtificial
SequenceSequence of C3Basic3 includes sequences from ADP-ribosyl
tranferase C3 (C. botulinum) and a sequence encoding a reverse
HIV-1 Tat amino acid sequence and a Histidine tag 34gga tcc tct aga
gtc gac ctg cag gca tgc aat gct tat tcc att aat 48Gly Ser Ser Arg
Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn1 5 10 15caa aag gct
tat tca aat act tac cag gag ttt act aat att gat caa 96Gln Lys Ala
Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln 20 25 30gca aaa
gct tgg ggt aat gct cag tat aaa aag tat gga cta agc aaa 144Ala Lys
Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 35 40 45tca
gaa aaa gaa gct ata gta tca tat act aaa agc gct agt gaa ata 192Ser
Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55
60aat gga aag cta aga caa aat aag gga gtt atc aat gga ttt cct tca
240Asn Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro
Ser65 70 75 80aat tta ata aaa caa gtt gaa ctt tta gat aaa tct ttt
aat aaa atg 288Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe
Asn Lys Met 85 90 95aag acc cct gaa aat att atg tta ttt aga ggc gac
gac cct gct tat 336Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp
Asp Pro Ala Tyr 100 105 110tta gga aca gaa ttt caa aac act ctt ctt
aat tca aat ggt aca att 384Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu
Asn Ser Asn Gly Thr Ile 115 120 125aat aaa acg gct ttt gaa aag gct
aaa gct aag ttt tta aat aaa gat 432Asn Lys Thr Ala Phe Glu Lys Ala
Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140aga ctt gaa tat gga tat
att agt act tca tta atg aat gtt tct caa 480Arg Leu Glu Tyr Gly Tyr
Ile Ser Thr Ser Leu Met Asn Val Ser Gln145 150 155 160ttt gca gga
aga cca att att aca aaa ttt aaa gta gca aaa ggc tca 528Phe Ala Gly
Arg Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly Ser 165 170 175aag
gca gga tat att gac cct att agt gct ttt cag gga caa ctt gaa 576Lys
Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185
190atg ttg ctt cct aga cat agt act tat cat ata gac gat atg aga ttg
624Met Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu
195 200 205tct tct gat ggt aaa caa ata ata att aca gca aca atg atg
ggc aca 672Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met
Gly Thr 210 215 220gct atc aat cct aaa gaa ttc aga agg aaa caa aga
aga aaa aga aga 720Ala Ile Asn Pro Lys Glu Phe Arg Arg Lys Gln Arg
Arg Lys Arg Arg225 230 235 240cac cac cac cac cac cac gtc gac tcg
agc ggc cgc atc gtg act gac 768His His His His His His Val Asp Ser
Ser Gly Arg Ile Val Thr Asp 245 250 255tga 77135256PRTArtificial
SequenceSequence of C3Basic3 includes sequences from ADP-ribosyl
tranferase C3 (C. botulinum) and a sequence encoding a reverse
HIV-1 Tat amino acid sequence and a Histidine tag 35Gly Ser Ser Arg
Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn1 5 10 15Gln Lys Ala
Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln 20 25 30Ala Lys
Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 35 40 45Ser
Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55
60Asn Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser65
70 75 80Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys
Met 85 90 95Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro
Ala Tyr 100 105 110Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser
Asn Gly Thr Ile 115 120 125Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala
Lys Phe Leu Asn Lys Asp 130 135 140Arg Leu Glu Tyr Gly Tyr Ile Ser
Thr Ser Leu Met Asn Val Ser Gln145 150 155 160Phe Ala Gly Arg Pro
Ile Ile Thr Lys Phe Lys Val Ala Lys Gly Ser 165 170 175Lys Ala Gly
Tyr Ile Asp Pro Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185 190Met
Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu 195 200
205Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr
210 215 220Ala Ile Asn Pro Lys Glu Phe Arg Arg Lys Gln Arg Arg Lys
Arg Arg225 230 235 240His His His His His His Val Asp Ser Ser Gly
Arg Ile Val Thr Asp 245 250 25536887DNAArtificial SequenceSequence
of C3APLT includes sequences from ADP-ribosyl transferase C3
(Clostridium botulinum) and a sequence encoding a proline rich
region. 36gga tcc tct aga gtc gac ctg cag gca tgc aat gct tat tcc
att aat 48Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser
Ile Asn1 5 10 15caa aag gct tat tca aat act tac cag gag ttt act aat
att gat caa 96Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn
Ile Asp Gln 20 25 30gca aaa gct tgg ggt aat gct cag tat aaa aag tat
gga cta agc aaa 144Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr
Gly Leu Ser Lys 35 40 45tca gaa aaa gaa gct ata gta tca tat act aaa
agc gct agt gaa ata 192Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 50 55 60aat gga aag cta aga caa aat aag gga gtt
atc aat gga ttt cct tca 240Asn Gly Lys Leu Arg Gln Asn Lys Gly Val
Ile Asn Gly Phe Pro Ser65 70 75 80aat tta ata aaa caa gtt gaa ctt
tta gat aaa tct ttt aat aaa atg 288Asn Leu Ile Lys Gln Val Glu Leu
Leu Asp Lys Ser Phe Asn Lys Met 85 90 95aag acc cct gaa aat att atg
tta ttt aga ggc gac gac cct gct tat 336Lys Thr Pro Glu Asn Ile Met
Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110tta gga aca gaa ttt
caa aac act ctt ctt aat tca aat ggt aca att 384Leu Gly Thr Glu Phe
Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125aat aaa acg
gct ttt gaa aag gct aaa gct aag ttt tta aat aaa gat 432Asn Lys Thr
Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140aga
ctt gaa tat gga tat att agt act tca tta atg aat gtt tct caa 480Arg
Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln145 150
155 160ttt gca gga aga cca att att aca aaa ttt aaa gta gca aaa ggc
tca 528Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly
Ser 165 170 175aag gca gga tat att gac cct att agt gct ttt gca gga
caa ctt gaa 576Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Ala Gly
Gln Leu Glu 180 185 190atg ttg ctt cct aga cat agt act tat cat ata
gac gat atg aga ttg 624Met Leu Leu Pro Arg His Ser Thr Tyr His Ile
Asp Asp Met Arg Leu 195 200 205tct tct gat ggt aaa caa ata ata att
aca gca aca atg atg ggc aca 672Ser Ser Asp Gly Lys Gln Ile Ile Ile
Thr Ala Thr Met Met Gly Thr 210 215 220gct atc aat cct aaa gaa ttc
gtg atg aat ccc gca aac gcg caa ggc 720Ala Ile Asn Pro Lys Glu Phe
Val Met Asn Pro Ala Asn Ala Gln Gly225
230 235 240aga cat aca ccc ggt acc aga ctc tag agctagagaa
ggagtttcac 767Arg His Thr Pro Gly Thr Arg Leu 245ttcaatcgct
acttgacccg tcggcgaagg atcgagatcg cccacgccct gtgcctcacg
827gagcgccaga taaagatttg gttccagaat cggcgcatga agtggaagaa
ggagaactga 88737248PRTArtificial SequenceSequence of C3APLT
includes sequences from ADP-ribosyl transferase C3 (Clostridium
botulinum) and a sequence encoding a proline rich region. 37Gly Ser
Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn1 5 10 15Gln
Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln 20 25
30Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys
35 40 45Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu
Ile 50 55 60Asn Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe
Pro Ser65 70 75 80Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser
Phe Asn Lys Met 85 90 95Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly
Asp Asp Pro Ala Tyr 100 105 110Leu Gly Thr Glu Phe Gln Asn Thr Leu
Leu Asn Ser Asn Gly Thr Ile 115 120 125Asn Lys Thr Ala Phe Glu Lys
Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140Arg Leu Glu Tyr Gly
Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln145 150 155 160Phe Ala
Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly Ser 165 170
175Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Ala Gly Gln Leu Glu
180 185 190Met Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met
Arg Leu 195 200 205Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr
Met Met Gly Thr 210 215 220Ala Ile Asn Pro Lys Glu Phe Val Met Asn
Pro Ala Asn Ala Gln Gly225 230 235 240Arg His Thr Pro Gly Thr Arg
Leu 2453837DNAArtificial SequenceOligonucleotide used in the
cloning of C3APLT in pET vector 38ggatctggtt ccgcgtcata tgtctagagt
cgacctg 373932DNAArtificial SequenceOligonucleotide used in the
cloning of C3APLT in pET vector 39cgcggatcca ttagttctcc ttcttccact
tc 324024DNAArtificial SequenceOligonucleotide used in the
sequencing of C3APLT 40aaattaatac gactcactat aggg
244119DNAArtificial SequenceOligonucleotide used in the sequencing
of C3APLT 41gctagttatt gctcagcgg 1942888DNAArtificial
SequenceSequence of C3APLT in a pET vector includes sequences from
ADP-ribosyl transferase C3 (Clostridium botulinum) and a sequence
encoding a proline rich region. 42atg tct aga gtc gca ctg cag gca
tgc aat gct tat tcc att aat caa 48Met Ser Arg Val Ala Leu Gln Ala
Cys Asn Ala Tyr Ser Ile Asn Gln1 5 10 15aag gct tat tca aat act tac
cag gag ttt act aat att gat caa gca 96Lys Ala Tyr Ser Asn Thr Tyr
Gln Glu Phe Thr Asn Ile Asp Gln Ala 20 25 30aaa gct tgg ggt aat gct
cag tat aaa aag tat gga cta agc aaa tca 144Lys Ala Trp Gly Asn Ala
Gln Tyr Lys Lys Tyr Gly Leu Ser Lys Ser 35 40 45gaa aaa gaa gct ata
gta tca tat act aaa agc gct agt gaa ata aat 192Glu Lys Glu Ala Ile
Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile Asn 50 55 60gga aag cta aga
caa aat aag gga gtt atc aat gga ttt cct tca aat 240Gly Lys Leu Arg
Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser Asn65 70 75 80tta ata
aaa caa gtt gaa ctt tta gat aaa tct ttt aat aaa atg aag 288Leu Ile
Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met Lys 85 90 95acc
cct gaa aat att atg tta ttt aga ggc gac gac cct gct tat tta 336Thr
Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr Leu 100 105
110gga aca gaa ttt caa aac act ctt ctt aat tca aat ggt aca att aat
384Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile Asn
115 120 125aaa acg gct ttt gaa aag gct aaa gct aag ttt tta aat aaa
gat aga 432Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys
Asp Arg 130 135 140ctt gaa tat gga tat att agt act tca tta atg aat
gtt tct caa ttt 480Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn
Val Ser Gln Phe145 150 155 160gca gga aga cca att att aca aaa ttt
aaa gta gca aaa ggc tca aag 528Ala Gly Arg Pro Ile Ile Thr Lys Phe
Lys Val Ala Lys Gly Ser Lys 165 170 175gca gga tat att gac cct att
agt gct ttt gca gga caa ctt gaa atg 576Ala Gly Tyr Ile Asp Pro Ile
Ser Ala Phe Ala Gly Gln Leu Glu Met 180 185 190ttg ctt cct aga cat
agt act tat cat ata gac gat atg aga ttg tct 624Leu Leu Pro Arg His
Ser Thr Tyr His Ile Asp Asp Met Arg Leu Ser 195 200 205tct gat ggt
aaa caa ata ata att aca gca aca atg atg ggc aca gct 672Ser Asp Gly
Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr Ala 210 215 220atc
aat cct aaa gaa ttc gtg atg aat ccc gca aac gcg caa ggc aga 720Ile
Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg225 230
235 240cat aca ccc ggt acc aga ctc tag agctagagaa ggagtttcac
ttcaatcgct 774His Thr Pro Gly Thr Arg Leu 245acttgacccg tcggcgaagg
atcgagatcg cccacgccct gtgcctcacg gagcgccaga 834taaagatttg
gttccagaat cggcgcatga agtggaagaa ggaggactaa ctga
88843247PRTArtificial SequenceSequence of C3APLT in a pET vector
includes sequences from ADP-ribosyl transferase C3 (Clostridium
botulinum) and a sequence encoding a proline rich region. 43Met Ser
Arg Val Ala Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn Gln1 5 10 15Lys
Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln Ala 20 25
30Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys Ser
35 40 45Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile
Asn 50 55 60Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro
Ser Asn65 70 75 80Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe
Asn Lys Met Lys 85 90 95Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp
Asp Pro Ala Tyr Leu 100 105 110Gly Thr Glu Phe Gln Asn Thr Leu Leu
Asn Ser Asn Gly Thr Ile Asn 115 120 125Lys Thr Ala Phe Glu Lys Ala
Lys Ala Lys Phe Leu Asn Lys Asp Arg 130 135 140Leu Glu Tyr Gly Tyr
Ile Ser Thr Ser Leu Met Asn Val Ser Gln Phe145 150 155 160Ala Gly
Arg Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly Ser Lys 165 170
175Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Ala Gly Gln Leu Glu Met
180 185 190Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg
Leu Ser 195 200 205Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met
Met Gly Thr Ala 210 215 220Ile Asn Pro Lys Glu Phe Val Met Asn Pro
Ala Asn Ala Gln Gly Arg225 230 235 240His Thr Pro Gly Thr Arg Leu
2454464PRTArtificial SequenceAmino acid sequence of Antennapedia
from C3APL 44Val Met Glu Ser Arg Lys Arg Ala Arg Gln Thr Tyr Thr
Arg Tyr Gln1 5 10 15Thr Leu Glu Leu Glu Lys Glu Phe His Phe Asn Arg
Tyr Leu Thr Arg 20 25 30Arg Arg Arg Ile Glu Ile Ala His Ala Leu Cys
Leu Thr Glu Arg Gln 35 40 45Ile Lys Ile Trp Phe Gln Asn Arg Arg Met
Lys Trp Lys Lys Glu Asn 50 55 604519PRTArtificial SequenceAmino
acid sequence of Antennapedia from C3APS 45Arg Gln Ile Lys Ile Trp
Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10 15Val Asp
Ser4660PRTArtificial SequenceAmino acid sequence of HIV-1 Tat from
C3-TL 46Lys His Pro Gly Ser Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr
Cys1 5 10 15Lys Lys Cys Cys Phe His Cys Gln Val Cys Phe Ile Thr Lys
Ala Leu 20 25 30Gly Ile Ser Tyr Gly Arg Lys Arg Arg Gln Arg Arg Arg
Ala His Gln 35 40 45Asn Ser Gln Thr His Gln Ala Ser Leu Ser Lys Gln
50 55 604720PRTArtificial SequenceAmino acid sequence of HIV-1 Tat
from C3-TS 47Tyr Gly Ala Lys Lys Arg Arg Gln Arg Arg Arg Val Asp
Ser Ser Gly1 5 10 15Pro His Arg Asp 204817PRTArtificial
SequenceAmino acid sequence of the proline rich region of C3APLT
48Val Met Asn Pro Ala Asn Ala Gln Gly Arg His Thr Pro Gly Thr Arg1
5 10 15Leu4910PRTArtificial SequenceAmino acid sequence fused to C3
protein to created C3 Tat-short 49Tyr Gly Arg Lys Arg Arg Gln Arg
Arg Arg1 5 10508PRTArtificial SequenceReverse sequence of Tat amino
acids fused to C3 protein to created C3Basic3 50Arg Arg Gln Arg Arg
Lys Lys Arg1 55112PRTArtificial Sequencetransport peptide rich in
Proline 51Ala Ala Val Leu Leu Pro Val Leu Leu Ala Ala Pro1 5
105224PRTArtificial SequenceSperm fertiline alpha peptide 52His Pro
Ile Gln Ile Ala Ala Phe Leu Ala Arg Ile Pro Pro Ile Ser1 5 10 15Ser
Ile Gly Thr Cys Ile Leu Lys20539PRTArtificial SequenceAmino acid
sequence from the C3Basic3 53Arg Arg Lys Gln Arg Arg Lys Arg Arg1
554744DNAArtificial SequenceSequence of C3-07Q189A 54atgtctagag
tcgacctgca ggcatgcaat gcttattcca ttaatcaaaa ggcttattca 60aatacttacc
aggagtttac taatattgat caagcaaaag cttggggtaa tgctcagtat
120aaaaagtatg gactaagcaa atcagaaaaa gaagctatag tatcatatac
taaaagcgct 180agtgaaataa atggaaagct aagacaaaat aagggagtta
tcaatggatt tccttcaaat 240ttaataaaac aagttgaact tttagataaa
tcttttaata aaatgaagac ccctgaaaat 300attatgttat ttagaggcga
cgaccctgct tatttaggaa cagaatttca aaacactctt 360cttaattcaa
atggtacaat taataaaacg gcttttgaaa aggctaaagc taagttttta
420aataaagata gacttgaata tggatatatt agtacttcat taatgaatgt
ttctcaattt 480gcaggaagac caattattac aaaatttaaa gtagcaaaag
gctcaaaggc aggatatatt 540gaccctatta gtgcttttgc aggagcactt
gaaatgttgc ttcctagaca tagtacttat 600catatagacg atatgagatt
gtcttctgat ggtaaacaaa taataattac agcaacaatg 660atgggcacag
ctatcaatcc taaagaattc gtgatgaatc ccgcaaacgc gcaaggcaga
720catacacccg gtaccagact ctag 74455247PRTArtificial SequenceAmino
acid sequence of C3-07Q189A 55Met Ser Arg Val Asp Leu Gln Ala Cys
Asn Ala Tyr Ser Ile Asn Gln1 5 10 15Lys Ala Tyr Ser Asn Thr Tyr Gln
Glu Phe Thr Asn Ile Asp Gln Ala 20 25 30Lys Ala Trp Gly Asn Ala Gln
Tyr Lys Lys Tyr Gly Leu Ser Lys Ser 35 40 45Glu Lys Glu Ala Ile Val
Ser Tyr Thr Lys Ser Ala Ser Glu Ile Asn 50 55 60Gly Lys Leu Arg Gln
Asn Lys Gly Val Ile Asn Gly Phe Pro Ser Asn65 70 75 80Leu Ile Lys
Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met Lys 85 90 95Thr Pro
Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr Leu 100 105
110Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile Asn
115 120 125Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys
Asp Arg 130 135 140Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn
Val Ser Gln Phe145 150 155 160Ala Gly Arg Pro Ile Ile Thr Gln Phe
Lys Val Ala Lys Gly Ser Lys 165 170 175Ala Gly Tyr Ile Asp Pro Ile
Ser Ala Phe Gln Gly Ala Leu Glu Met 180 185 190Leu Leu Pro Arg His
Ser Thr Tyr His Ile Asp Asp Met Arg Leu Ser 195 200 205Ser Asp Gly
Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr Ala 210 215 220Ile
Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg225 230
235 240His Thr Pro Gly Thr Arg Leu 24556783DNAArtificial
SequenceSequence of BA-05 56ggatcctcta gagtcgacct gcaggcatgc
aatgcttatt ccattaatca aaaggcttat 60tcaaatactt accaggagtt tactaatatt
gatcaagcaa aagcttgggg taatgctcag 120tataaaaagt atggactaag
caaatcagaa aaagaagcta tagtatcata tactaaaagc 180gctagtgaaa
taaatggaaa gctaagacaa aataagggag ttatcaatgg atttccttca
240aatttaataa aacaagttga acttttagat aaatctttta ataaaatgaa
gacccctgaa 300aatattatgt tatttagagg cgacgaccct gcttatttag
gaacagaatt tcaaaacact 360cttcttaatt caaatggtac aattaataaa
acggcttttg aaaaggctaa agctaagttt 420ttaaataaag atagacttga
atatggatat attagtactt cattaatgaa tgtttctcaa 480tttgcaggaa
gaccaattat tacaaaattt aaagtagcaa aaggctcaaa ggcaggatat
540attgacccta ttagtgcttt tgcaggacaa cttgaaatgt tgcttcctag
acatagtact 600tatcatatag acgatatgag attgtcttct gatggtaaac
aaataataat tacagcaaca 660atgatgggca cagctatcaa tcctaaagaa
ttcgtgatga atcccgcaaa cgcgcaaggc 720agacatacac ccggtaccag
actctagagc tagagaagga gtttcacttc aatcgctact 780tga
78357247PRTArtificial SequenceAmino acid sequence of pET9a-BA-07
57Met Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn Gln1
5 10 15Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln
Ala 20 25 30Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser
Lys Ser 35 40 45Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser
Glu Ile Asn 50 55 60Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly
Phe Pro Ser Asn65 70 75 80Leu Ile Lys Gln Val Glu Leu Leu Asp Lys
Ser Phe Asn Lys Met Lys 85 90 95Thr Pro Glu Asn Ile Met Leu Phe Arg
Gly Asp Asp Pro Ala Tyr Leu 100 105 110Gly Thr Glu Phe Gln Asn Thr
Leu Leu Asn Ser Asn Gly Thr Ile Asn 115 120 125Lys Thr Ala Phe Glu
Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp Arg 130 135 140Leu Glu Tyr
Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln Phe145 150 155
160Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly Ser Lys
165 170 175Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Ala Gly Gln Leu
Glu Met 180 185 190Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp
Met Arg Leu Ser 195 200 205Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala
Thr Met Met Gly Thr Ala 210 215 220Ile Asn Pro Lys Glu Phe Val Met
Asn Pro Ala Asn Ala Gln Gly Arg225 230 235 240His Thr Pro Gly Thr
Arg Leu 2455835DNAArtificial Sequenceprimer 58cctaaagaat tcgtgatgaa
tcccgcaaac gcgca 355935DNAArtificial Sequenceprimer 59tgcgcgtttg
cgggattcat cacgaattct ttagg 35
* * * * *